Oligonucleotide-ligand conjugates and process for their preparation

ABSTRACT

The present invention relates to ligand conjugates of oligonucleotides (e.g., iRNA agents) and methods for their preparation. The ligands are derived primarily from monosaccharides These conjugates are useful for the in vivo delivery of oligonucleotides.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/845,279, filed Jul. 11, 2013, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to ligand conjugates of oligonucleotides(e.g., iRNA agents) and methods for their preparation. The ligands arederived primarily from monosaccharides. These conjugates are useful forthe in vivo delivery of oligonucleotides.

BACKGROUND OF THE INVENTION

Efficient delivery to cells in vivo requires specific targeting andsubstantial protection from the extracellular environment, particularlyscrum proteins. One method of achieving specific targeting is toconjugate a targeting moiety to the iRNA agent. The targeting moietyhelps in targeting the iRNA agent to the required target site. One way atargeting moiety can improve delivery is by receptor mediatedendocytotic activity. This mechanism of uptake involves the movement ofiRNA agent bound to membrane receptors into the interior of an area thatis enveloped by the membrane via invagination of the membrane structureor by fusion of the delivery system with the cell membrane. This processis initiated via activation of a cell-surface or membrane receptorfollowing binding of a specific ligand to the receptor. Manyreceptor-mediated endocytotic systems are known and have been studied,including those that recognize sugars such as galactose, mannose,mannose-6-phosphate, peptides and proteins such as transferrin,asialoglycoprotein, vitamin B12, insulin and epidermal growth factor(EGF). The Asialoglycoprotein receptor (ASGP-R) is a high capacityreceptor, which is highly abundant on hepatocytes. The ASGP-R shows a50-fold higher affinity for N-Acetyl-D-Galactosylamine (GalNAc) thanD-Gal. Previous work has shown that multivalency is required to achievenM affinity, while spacing among sugars is also crucial.

Recently, certain carbohydrate conjugates have been shown to be avaluable delivery alternatively to liposomes for siRNA delivery.

SUMMARY OF THE INVENTION

The present invention relates to ligand conjugates of oligonucleotidesor other biologically active agents which have one or more advantageousproperties, such as improved delivery of the oligonucleotide or otherbiologically active agents, lower manufacturing costs or fewermanufacturing issues, or better chemical stability. These conjugatesprovide effective delivery of oligonucleotides and other biologicallyactive agents.

In one embodiment, the present invention relates to a ligand (e.g.,carbohydrate) conjugate of an oligonucleotide (e.g., an iRNA agent) orother biologically active agent of the formula I:

wherein

the Oligonucleotide is an oligonucleotide, such as an siRNA, microRNA,antimiR, antagomir, microRNA mimic, decoy, immune stimulatory,G-quadruplex, splice altering, ssRNA, antisense, aptamer, stem-loop RNAor DNA or one of the two strands of any double stranded RNA or DNA ordouble stranded shortener RNA or DNA (e.g, siRNA);

the Biologically Active Agent is any biologically active agent;

the Linker is a linking group between the Ligand(s) and theOligonucleotide or other biologically active agent, where the Linker maybe selected from the linking groups in Table 1 or 1A; and

the Ligand(s) are sugar-derived, where (i) the Ligand(s) may be attachedto the same or different atoms in the Linker, (ii) the conjugatecontains from 1 to 12 Ligands (preferably 1 to 5 or 1 to 3 Ligands), and(iii) the Ligand(s) may be selected from

-   -   (a) the Ligands in Table 2 or 2A,    -   (b) R²—(R³)_(k), where        -   R² is absent (in which case k=1) or a spacer (also referred            to as a ligand backbone) having two or more sites of            attachment for the R³ groups.        -   R³ is a targeting monomer selected from Table 3, and k is 1            to 6 (preferably 1 to 5 or 1 to 3), each R³ may be attached            to the same or different atoms in R²; and    -   (c) the Ligands in Table 4 or 4A.

The conjugate includes at least one Linker from Table 1 or 1A or fromthe examples, one Ligand from Table 2, 2A, 4, or 4A, or one targetingmonomer from Table 3 or 3A. For example, the nucleoside linkersdescribed in the examples can be used as the Linker. In one embodiment,the conjugate includes (i) at least one Linker from Table 1 or 1A orfrom the examples, (ii) one Ligand from Table 2, 2A, 4, or 4A, and (iii)one targeting monomer from Table 3 or 3A.

R² can be an amino acid-, polypeptide- (e.g., a dipeptide ortripeptide), heteroaryl- (e.g., a triazole), or sugar-containing group.In one preferred embodiment, each R³ group is attached via an amide,ether, or amino group to R². In one embodiment, R³ is attached via anamide group. Each entry in Tables 2, 2A, 4, and 4A shows a spacer boundto at least one targeting monomer. The spacers are bound to thetargeting monomers either through a heteroatom (which is at a terminusof the spacer), such as a nitrogen atom, or at the anomeric carbon tothe sugar group of a targeting monomer (as shown below). The heteroatomattachment point in the structures shown in Tables 2, 2A, 4, and 4A isthe first nitrogen atom when walking along the chain from the sugargroup (left side of the ligand) to the remainder of the ligand (rightside). The spacers for two ligands in Table 2A are shown in the tablebelow (the arrows indicate the points of attachment for the targetingmonomers R³, and the right side of the spacer is attached to theLinker). Suitable spacers are also shown in Table 5 below.

Ligand from Table 2A

Spacer Component (R²) of Ligand

The Oligonucleotide is preferably attached to the Linker through (i) the3′ or 5′-terminal of the oligonucleotide, (ii) one or more sugarmoieties of the nucleoside present in the oligonucleotide independent ofposition, or (iii) one or more base moieties of the nucleoside presentindependent of position.

In one embodiment, the Ligand is conjugated to one of e two strands of adouble stranded siRNA via a Linker.

In one embodiment, the Ligand(s) target the asialoglycoprotein receptor(ASGPR). In another embodiment, the Ligand(s) target the liver, such asthe parenchymal cells of the liver. The Ligand(s) may be an unmodifiedor modified monosaccharide, disaccharide, trisaccharide,tetrasaccharide, or higher polysaccharide.

The oligonucleotide can be attached to the Linker via a cleavable group(e.g., phosphate, phosphorothiate, or amide) or a non-cleavable group(e.g., ether, carbamate, or C—C(e.g., a bond between two carbon atoms orCH₂—CH₂—)). As described herein, the cleavable or non-cleavable group iswithin the Oligonucleotide of Formula I.

In one embodiment, the -Linker-Ligand is not L96 (shown in theexamples).

In the formulas of the conjugates described herein (such as Formula(1)), the oligonucleotide or other biologically active agent can bereplaced by a component of a lipid nanoparticle (LNP) (such as aPEG-lipid or cationic lipid) or a polymer. The conjugated LNP componentor conjugated polymer may be useful as delivery agents for facilitatingdelivery of a biologically active agent to a target site.

TABLE 1 Linker Groups^(a,b) The Linkers below are shown with theprotecting group DMTr. When conjugated, the DMTr group is removed andthe adjacent oxygen atom is the site of attachment of the Linker to theoligonucleotide (e.g., to a cleavable group of the oligonucleotide). Thesquiggly line is the point of attachment for the Ligand. X can behydrogen, a leaving group, —OH, or —NH₂. When the Linker group isincorporated into an intermediate compound useful for preparing aconjugate of the present invention, X can be a reactive phosphoramidite(e.g.,

compatible with solid phase oligonucleotide synthesis and deprotectionor attached to a solid support (e.g.,

that enables solid phase oligonucleotide synthesis.

When conjuagated, the DMTr group is removed and the adjacent oxygen isthe site of attachment of the Linker to the oligonucleotide (e.g., via acleavable group such as a phosphate or phosphorothioate) or otherbiologically active agent ^(a )

 indicates the site of attachment of the Ligand. ^(b)Each structurerepresents chirally pure or racemic isomers when one or more asymmetriccenters are present.

TABLE 1A Linker Groups The Linkers below in Table 1A arc shown with oneor more oligonucleotides attached to them. It will be understood tothose skilled in the art that the Linker is the chemical moiety withoutthe oligonucleotide(s). The squiggly line is the point of attachment forthe Ligand. X can be hydrogen, a leaving group, —OH, or —NH₂. When theLinker group is incorporated into an intermediate compound useful forpreparing a conjugate of the present invention. X can be a reactivephosphoramidite (e.g.,

compatible with solid phase oligonucleotide synthesis and deprotectionor attached to a solid support (e.g.,

that enables solid phase oligonucleotide synthesis. The phrase“oligonucleotide/nucleotide” is meant to refer to a single nucleotide oran oligonucleotide.

 indicates site of attachment of the ligand; Each structure representschirally pure or racemic isomers when one or more asymmetric centerspresent. The linkage between oligonucleotide/nucleotide and theconjugate moiety is phosphate or phosphorothioate. m and p areindependently 1-8 (e.g., 1-4).

TABLE 2 Ligands^(a,b,d)

^(a)Q = O, S, CH₂: Z = —CONH—, —NHCO—, —OC(O)NH—, or —NHC(O)O—; R is thepoint of attachment to a Linker; R′ = Ac, COCF₃ or any amine protectinggroup compatible with oligonucleotide (RNA/DNA) synthesis anddeprotection conditions. ^(b)each of the variables l, m, n, p, q, and rindepentdently ranges from about 0 to about 10. ^(d)Each structurerepresents chirally pure or racemic isomers when one or more asymmetriccenters are present.

TABLE 2A Ligands^(a,b,c,d)

^(a)Q is O, S, or CH₂; and Z is —CONH—, —NHCO—, —OC(O)NH—, or —NHC(O)O—.^(b)each of the variables l, m, n, p, q, and r independently ranges fromabout 0 to about 10. ^(c)

 indicates site of attachment of the Ligand to the Linker. ^(d)Eachstructure represents chirally pure or racemic isomers when one or moreasymmetric centers are present. The variable R, unless otherwisespecified, is OH or NHAc.

TABLE 3^(a) Targeting Monomers (R³)

^(a)These groups are functional monomers to conjugate to amino linkedoligonucleotides. Each structure represents chirally pure or racemicisomers when one or more asymmetric centers are present. → indicate siteof conjugation. The variable X, unless otherwise specified, is O, S, C,or NHCO. The variable n, unless otherwise specified, is 1-8 (e.g., 1-4).

TABLE 3A Targeting Monomers (R³) Table 1. ASGPR Ligand Mimics^(a)

The variable R, unless otherwise specified, is OH or NHAc. The variablen, unless otherwise specified, is 1-8 (for example, 1-4). The variableR′ , unless otherwise specified, is H or Ac. The variable X, unlessotherwise specified, is O, S, C, or NHCO. The variable Z, unlessotherwise specified, is NH, NHAc, S, or O.

Ligands

The ligands can also be selected from the two generic formula below

where the arrow indicates the point of attachment to the oligonucleotideconjugate (i.e., the ligand is attached through its carbonyl group).Intermediates useful for introducing the ligand include the compoundsshown above. In the formulas above, the variables have the definitionsprovided below.

R⁶ is H or Ac:

R⁷ is OH or NHR⁹;

R⁸ is Ac or R⁹; where at least one of R⁷ and R⁸ is a nitrogen containingmoiety;

R⁹ is

Q¹ is H, C₁-C₄ alkyl,

Q² is H or C₁-C₄ alkyl;

X is H or Me;

Y is H, Ac, or COCF₃; and

n is 1 to 8 (e.g., 1 to 4).

The ligands may have the formulas shown in Table 4 or 4A below.

TABLE 4^(b)

X = H, Me Y = H, Ac, COCF₃ Q1 = H, CH₃, Et, nPr, isoPr, nBu, isoBu

Q2 = H, Me, Et, nPr, isoPr, isoBu, nBu

n is 1 to 8, e.g., 1 to 4 ^(b)Each structure represents chirally pure orracemic isomers when one or more asymmetric centers are present. Thearrow indicates the point of attachment to the Linker.

TABLE 4A^(b)

X = H, Me Y = H, Ac, COCF₃ Q1 = H, CH₃, Et, nPr, isoPr, nBu, isoBu

Q2 H, Me, Et, nPr, isoPr, isoBu, nBu

n is 1 to 8, e.g., 1 to 4 ^(b)Each structure represents chirally pure orracemic isomers when one or more asymmetric centers present R = OH orNHAc in all occurrence The squiggly linke indicates the point ofattachment to the Linker.

TABLE 5 Spacers (R²)

  R′ = H or Ac

  (the Linker attached through the NH group at the 6-position of thesugar shown above.)

  (the Linker attached through the NH group at the 6-position of thesugar shown above.)

  R′ = H, Ac

All the squiggly lines (except for the right most squiggly lines) arethe points of attachment for the targeting monomers R³. The right mostsquiggly line is the point of attachment of the Linker. Each of thevariables m, n, q, and r independently ranges from about 0 to about 10.

In tables 2, 2A, 3, 3A, 4, and 4A and other formulas which include oneor more —OAc substituents on the sugar moiety, the compounds of thepresent invention include the identical compounds containing —OHsubstituents at the one or more —OAc positions shown. In general, theacetyl (Ac) group acts as protecting group for the hydroxyl moiety.Accordingly, it will be understood by those skilled in the art that thecorresponding hydroxy compounds are within the scope of the presentinvention and are intended to be used in the final conjugates with anoligonucleotide or other biologically active agent.

In addition, the compounds of the present invention also include thosein which any —NHAc substituent on the sugar moiety is replaced with ahydroxy group (e.g., where the NHAc group at the 2-position of the sugarmoiety is replaced with a 2-OH group). In one embodiment, in addition tothe replacement of the —NHAc group(s) with hydroxy group(s), any —OAcsubstituents on the sugar moiety are also replaced with hydroxygroup(s).

In one embodiment, the Ligand(s) are selected from:

where

A is the attachment point to the Linker, and can represent a bond or achemical linkage group (e.g., an amide, carbamate, urea, —C—N— (e.g.,—CH₂—NH— or —C(R^(a))(R^(b))—N(R^(c))— where R^(a), R^(b), and R^(c) areindependent selected from hydrogen, alkyl, and aryl), C═NH, ether,thioether, triazole, oxime, or hydrazine) which is attached to theLinker;

Y is any functional group (e.g., when used as a divalent group, it canbe —CONH—, —NHCO—, or S, or, e.g., when used as a monovalent group, itcan be OH, —SH, or halogen), —CH₂—, protecting group, or chemicallyinactive cap;

Q is OH, or any modification to the C6 position of the sugar describedherein; and

n is 1 to 6.

In yet another embodiment, the sugar moiety in any of the aforementionedligands or targeting monomers (such as in Tables 2, 2A, 3, 3A, 4, and4A) may be replaced with the sugar moiety of formula III below

wherein

each occurrence of R⁶ is independently as defined above (e.g., H or Ac);

R⁷ and R″ are independently selected from Z—R¹⁰, unsubstituted andsubstituted heteroaryl (e.g., a triazole or imidazole), —N₃, —CN, andsubstituted and unsubstituted acetylene;

each occurrence of Z is independently O, NH, or S;

each occurrence of R¹⁰ is independently H, unsubstituted or substitutedalkyl, unsubstituted acyl (e.g., —COCH₃), substituted acyl (e.g.,—COCF₃), —OC(O)OR¹¹, —NHC(O)OR¹¹, —NHC(O)NHR¹¹, or an amino acid; and

each occurrence of R¹¹ is independently H or unsubstituted orsubstituted alkyl,

with the proviso that R⁷ is not OH or —OAc when (i) R⁶ is H or (ii) R″is OH or NHAc.

Yet another embodiment is an intermediate compound of the formula

where

Ligand(s) and Linker are as defined above, and

X is

(where the sphere represents a solid support),

a leaving group, H, —OH, or —NH₂.These intermediates are useful for preparing the olignucleotide-ligandconjugates of the present invention.

Yet another embodiment is an intermediate compound of the formula IIIA

wherein

R⁵² is a bivalent, chemical group of 1 to 12 atoms in length;

Linker is defined as above;

each occurrence of R⁶ is independently as defined above (e.g., H or Ac);

R⁷ and R″ are independently selected from —Z—R¹⁰, unsubstituted andsubstituted heteroaryl (e.g., a triazole or imidazole), —N₃, —CN, andsubstituted and unsubstituted acetylene;

each occurrence of Z is independently O, NH, or S;

each occurrence of R¹⁰ is independently H, unsubstituted or substitutedalkyl, unsubstituted acyl (e.g., —COCH₃), substituted acyl (e.g.,—COCF₃), —OC(O)OR¹¹, —NHC(O)OR¹¹, —NHC(O)NHR¹¹, or an amino acid;

each occurrence of R¹¹ is independently H or unsubstituted orsubstituted alkyl; and

X is

(where the sphere represents a solid support),

a leaving group, H, —OH, or NH₂,

with the proviso that R⁷ is not —OH or —OAc when (i) R⁶ is H or (ii) R″is OH or NHAc.

In one preferred embodiment, the substitutions at the 3- and 4-positionsof the sugar group in formula III or IIIA are equatorial and axial,respectively.

In one embodiment, the sugar in formula III or IIIA is in an alphaconfiguration. In another embodiment, the sugar is in a betaconfiguration.

The present invention also includes compounds of formula IIIA where X isreplaced by an oligonucleotide or other biologically active agent asdescribed herein.

The same linkage or a combination of linkages described herein can beused for attaching two or more ligand/linker moieties to theoligonucleotide or other biologically active agent. For example, in oneembodiment, the invention relates to a conjugate of an oligonucleotide(e.g., an iRNA agent) or other biologically active agent of the formulaIV

wherein

the Ligand(s) and Linker are as defined above;

Oligonucleotide-1 and Oligonucleotide-2 have the same definition asOligonucleotide above;

Biologically Active Agent-1 and Biologically Active Agent-2 have thesame definition as Biologically Active Agent above; and

each Ligand may be the same or different and each Oligonucleotide may bethe same or different.

In formula IV, the Linker connects two segments of the oligonucleotide(oligonucleotide 1 and 2) via two linkages. Each segment of theoligonucleotide represents at least one nucleoside moiety.

Yet another embodiment is a conjugate of an oligonucleotide (e.g., anIRNA agent) or other biologically active agent of the formula V:

wherein

each Oligonucleotide (or Biologically Active Agent) and Ligand areindependently as defined herein;

each Linker can independently be any those described herein (e.g., inTable 1 or 1A) or can have the formula

where R⁴ is the site of attachment to the oligonucleotide (for example,via a cleavable group in the oligonucleotide), and the hydroxy group onthe hydroxyproline is the site of attachment for an additional-Linker-Ligand group; and

t ranges from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6).

In one preferred embodiment, t is 2.

In one preferred embodiment, the oligonucleotides in the conjugatesdescribed herein are attached to the Linker via a phosphate,phosphorothioate, or a combination thereof.

In one embodiment, the conjugate is an oligonucleotide conjugate havingthe formula:

wherein each R is independently a Ligand (such as those describedherein). In one preferred embodiment, the Ligands R are the same.

The present invention also relates to a ligand conjugate of anoligonucleotide where at least one nucleoside is conjugated to acarbohydrate-containing Ligand either (i) through the nucleobase of thenucleoside or (ii) at the 2′-position of the nucleoside.

One embodiment is a carbohydrate conjugate of an oligonucleotide,wherein at least one nucleoside in the oligonucleotide is conjugated toa carbohydrate-containing Ligand (e.g., a sugar-containing Ligand) via anitrogen atom in the nucleobase of the nucleoside. Any Ligand describedherein may be used. In one embodiment, the nucleoside in the conjugateis of the formula VI:

where

the 5′ and 3′ ends of the nucleoside in Formula VI are each attached toanother nucleoside of the oligonucleotide or to a terminus:

R⁶ is a nucleobase (e.g., uracil, cytosine, adenosine, or guanine) andoptionally has a nitrogen-containing moiety bound to the nucleobase;

R⁷ is a Linker, where R⁷ is bound to a nitrogen atom (e.g., an aminogroup) in R⁶;

each R⁸ is independently a Ligand. Each R⁸ may be attached to the sameor different atoms in the Linker R⁷. The ligand R⁸ can be, for example,—R²-R³ or the ligands in Tables 2, 2A, 4, and 4A

In one embodiment, R⁶ is uracil substituted at its 5-position with anamide group —C(O)NH—, where R⁷ is bound to R⁶ through the nitrogen atomof the amide group.

In another embodiment, R⁶ is cytosine substituted at its 5-position withan amide group —C(O)NH—, where R⁷ is bound to R⁶ through the nitrogenatom of the amide group.

Another embodiment is a carbohydrate conjugate of an oligonucleotide,wherein at least one nucleoside in the oligonucleotide is conjugated toa Ligand (e.g., sugar-containing Ligand) at its 2″-position. Any Liganddescribed herein may be used. In one embodiment, the nucleoside in theconjugate is of the formula VII:

where

the 5′ and 3′ ends of the nucleoside in Formula VII are each attached toanother nucleoside of the oligonucleotide or to a terminus;

R⁶ is a nucleobase;

R⁷ is a Linker:

each R⁸ is independently a Ligand. Each R⁸ may be attached to the sameor different atoms in the Linker R⁷. In one preferred embodiment, theoligonucleotide is attached to the Linker via a phosphate,phosphorothioate, or a combination thereof. For example, theoligonucleotide may be attached to a Linker at the 3′-end via aphosphate and/or a phosphorothioate at the 5′-end, or vice versa.

The ligand moiety (e.g., a carbohydrate moiety) facilitates delivery ofthe oligonucleotide to the target site. One way a ligand moiety canimprove delivery is by receptor mediated endocytotic activity. Withoutbeing bound by any particular theory, it is believed that this mechanismof uptake involves the movement of the oligonucleotide bound to membranereceptors into the interior of an area that is enveloped by the membranevia invagination of the membrane structure or by fusion of the deliverysystem with the cell membrane. This process is initiated via activationof a cell-surface or membrane receptor following binding of a specificligand to the receptor. Receptor-mediated endocytotic systems includethose that recognize sugars such as galactose. The ligand moietytherefore may include one or more monosaccharides, disaccharides,trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides,such as those described above. In one preferred embodiment, the ligandmoiety may be a moiety which is recognized by a human asialoglycoproteinreceptor (ASGPR), such as human asialoglycoprotein receptor 2 (ASGPR2).Such a carbohydrate moiety may, for instance, comprise a sugar (e.g.,galactose or N-acetyl-D-galactosylamine).

Yet another embodiment is an oligonucleotide in which two or morenucleotides each have a Linker-Ligand moiety. The Linker-Ligand moietiesin the oligonucleotide may be the same or different. In one embodiment,the first, third, and fifth nucleotides from the 5′ terminus are eachconjugated to a Linker-Ligand moiety. In another embodiment, the first,third, and fifth nucleotides from the 3′ terminus are each conjugated toa Linker-Ligand moiety. In yet another embodiment, the first, third, andfifth nucleotides from the 3′ and 5′ ends of the oligonucleotide areeach conjugated to a Linker-Ligand moiety.

Yet another embodiment is a method of formulating a therapeutic RNA bypreparing a conjugate of an IRNA agent of the present invention, where astrand of the IRNA agent comprises the therapeutic RNA.

Yet another embodiment is a method of delivering a therapeutic RNA to apatient in need thereof by administering to the patient a conjugate ofan IRNA agent of the present invention, where the a strand of the iRNAagent comprises the therapeutic RNA. Preferred routes of administrationinclude the subcutaneous and intravenous routes,

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing tranthyretin (TTR) protein levels 48 and144 hours after administration of TTR siRNA conjugates in mice relativeto control mice according to the procedure in Example 33.

FIG. 2 is a bar graph showing antithrombin 3 (AT3) protein levelsfollowing administration of AT3 siRNA conjugates in mice relative tocontrol mice according to the procedure in Example 34.

FIGS. 3A and 3B are bar graphs showing mTTR protein levels 72 hours(FIG. 3A) and 144 hours (FIG. 3B) following a single subcutaneous doseof conjugates 57727, 63189, 63192, 63190 and 63191 to mice according tothe procedure in Example 62.

FIG. 4 is a bar graph of the binding affinities (Ki) of the TTR siRNAconjugates 56718-56727, 56729 and 55727 in Example 42.

FIG. 5 is a graph (binding affinity curve) showing the medianfluorescence intensity (MA) at various concentrations for the TTR siRNAconjugates 56727, 56729 and 55727 in Example 42.

FIG. 6 is a graph showing the median fluorescence intensity (MFI) atvarious concentrations for the TTR siRNA conjugates 56721, 56722, 56723and 55727 in Example 42.

FIG. 7 is a graph showing the median fluorescence intensity (MFI) atvarious concentrations for the TTR siRNA conjugates 56724, 56725, 56726,56718, 56719, 56720 and 55727 in Example 42,

FIGS. 8 and 9 are graphs the median fluorescence intensity (MFI) atvarious concentrations for the TTR siRNA conjugates 56876, 66875, 56874,66878, 56880, 56879, 54944, 56877, 56881 and/or 56882 in Example 44.

FIG. 10 is a bar graph showing TTR protein levels 48 and 144 hours afteradministration of TTR siRNA conjugates 43527, 60126, 60138, 60128,60127, 60316, and 60123 (at 15 mg/kg and 5 mg/kg doses) in mice relativeto control mice as described in Example 45.

FIG. 11 is a bar graph showing AT3 protein levels followingadministration of AT3 siRNA conjugates 54944, 56881 and 58137 in micerelative to control mice as described in Example 46.

FIG. 12 is a bar graph showing mTTR protein Levels followingadministration of mTTR siRNA conjugates 55727, 58138 and 58139 in micerelative to control mice as described in Example 46.

FIG. 13 is a graph showing the median fluorescence intensity (MFI) atvarious concentrations for the TTR siRNA conjugates 61696, 61695, 61692,61694, 61697, 61693, 43527 and 61698 in Example 61.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “oligonucleotide” refers to a chemically modified or unmodifiednucleic acid molecule (RNA or DNA) having a length of less than about100 nucleotides (for example, less than about 50 nucleotides. Thenucleic acid can, for example, be (i) single stranded DNA or RNA, (ii)double stranded DNA or RNA, including double stranded DNA or RNA havinga hairpin loop, or (iii) DNA/RNA hybrids. Non-limiting examples ofdouble stranded RNA include siRNA (small interfering RNA). Singlestranded nucleic acids include, antisense oligonucleotides, ribozymes,microRNA, and triplex forming oligonucleotides. In one embodiment, theoligonucleotide has a length ranging from about 5 to about 50nucleotides (such as from about 10 to about 50 nucleotides). In anotherembodiment, the oligonucleotide has a length ranging from about 6 toabout 30 nucleotides, such as from about 15 to about 30 nucleotides. Inyet another embodiment, the oligonucleotide has a length ranging fromabout 18 to about 23 nucleotides.

The term “GalNAc” refers to N-acetyl-galactosamine.

The term “solid support,” as used herein denotes in particular anyparticle, bead, or surface upon which synthesis of an oligonucleotideoccurs. Solid supports which can be used in the different embodiments ofthe processes described herein can be selected for example frominorganic supports and organic supports. Inorganic supports arepreferably selected from silica gel and controlled pore glass (CPG).Organic supports are preferably selected from highly crosslinkedpolystyrene, Tentagel (grafted copolymers consisting of a lowcrosslinked polystyrene matrix on which polyethylene glycol (PEG or POE)is grafted), polyvinylacetate (PVA), Poros—a copolymer ofpolystyrene/divinyl benzene, aminopolyethyleneglycol and cellulose.Preferred solid supports amenable to this invention include those thatare hydrophobic. Preferred embodiments of the invention utilizepolystyrene based solid supports. Many other solid supports arecommercially available and amenable to the present invention.

The term “hydroxy protecting group,” as used herein, refers to a labilechemical moiety which protects a hydroxyl group against undesiredreactions during synthetic procedure(s). After the syntheticprocedure(s), the hydroxy protecting group may be selectively removed.Hydroxy protecting groups as known in the art are described generally inT. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis,3rd edition, John Wiley & Sons, New York (1999). Examples of hydroxylprotecting groups include, but are not limited to, benzyloxycarbonyl,4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl,isopropoxycarbonyl, diphenylmethoxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl) ethoxycarbonyl,2-furfuryloxycarbonyl, allyloxycarbonyl, acetyl, formyl, chloroacetyl,trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, methyl, t-butyl,2,2,2-trichloroethyl, 2-trimethylsilyl ethyl, 1,1-dimethyl-2-propenyl,3-methyl-3-butenyl, allyl, benzyl, para-methoxybenzyldiphenylmethyl,triphenylmethyl (trityl), tetrahydrofuryl, methoxymethylmethylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl,2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, para-toluenesulfonyl,trimethylsilyl, triethylsilyl, and triisopropylsilyl. Preferred hydroxylprotecting groups for the present invention are acetyl (Ac or —C(O)CH₃),benzoyl (Bz or —C(O)C₆H₅), and trimethylsilyl (TMS or —Si(CH₃)₃).

The term “amino protecting group,” as used herein, refers to a labilechemical moiety which protects an amino group against undesiredreactions during synthetic procedures. After the synthetic procedure(s),the amino protecting group as described herein may be selectivelyremoved. Amino protecting groups as known in the are described generallyin T, H. Greene and P. G. M. Wuts, Protective Groups in OrganicSynthesis, 3rd edition. John Wiley & Sons, New York (1999). Examples ofamino protecting groups include, but are not limited to, acetyl,t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl and benzyloxycarbonyl.

The term “carboxylic acid protecting group” refers to carboxylic acidprotecting groups employed to block or protect the carboxylic acidfunctionality while reactions involving other functional sites of thecompound are carried out. Such carboxy protecting groups may be notedfor their ease of cleavage by hydrolytic or by hydrogenolytic methods tothe corresponding carboxylic acid. Examples of carboxylic acid esterprotecting groups include, but are not limited to, methyl, tert-butyl,benzyl, 4-methoxybenzyl, C2-C6 alkanoyloxymethyl, 2-iodoethyl,4-nitrobenzyl, diphenylmethyl (benzhydryl), phenacyl, 4-halophenacyl,dimethylallyl, 2,2,2-trichloroethyl, tri(C1-C3 alkyl)silyl,succinimidomethyl and like ester forming moieties. In addition to esterprotection of carboxy groups, such groups can also be protected as themixed anhydride, such as that formed with acetyl chloride, propionylchloride, isobutyryl chloride and other acid chlorides in the presenceof a tertiary amine base. Other known carboxy protecting groups such asthose described by E. Haslam in Protective Groups in Organic Chemistry,supra, Chapter 5, are suitable. The ester forming protecting groups arepreferred.

In the foregoing definitions hydroxy and carboxy protecting groups arenot exhaustively defined. The function of such groups is to protect thereactive functional groups during the preparative steps and then to beremoved at some later point in time without disrupting the remainder ofthe molecule. Many protecting groups are known in the art, and the useof other protecting groups not specifically referred to hereinabove areequally applicable.

Suitable peptide coupling reagents include, but are not limited to, DCC(dicyclohexylcarbodiimide), DIC (diisopropylearbodiimide),di-p-toluoylcarbodiimide, BDP (1-benzotriazolediethylphosphate-1-cyclohexyl-3-(2-morpholinylethyl)carbodiimide), EDC(1-(3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuricfluoride, cyanuric chloride, TFFH (tetramethyl fluoroformamidiniumhexafluorophosphosphate), DPPA (diphenylphosphorazidate), BOP(benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate), HBTU(O-benzotriazol-1-yl-N,N,N′,N′-tetramethylitronium hexafluorophosphate),TBTU (O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumtetrafluoroborate), TSTU(O—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate),HATU(N-[(dimethylamino)-1-H-1,2,3-triazolo[4,5,6]-pyridin-1-ylmethylene]-N-methylmethanaminiumhexafluorophosphate N-oxide), BOP—Cl(bis(2-oxo-3-oxazolidinyl)phosphinic chloride). PyBOP((1-H-1,2,3-benzotriazol-1-yloxy)-tris(pyrrol idino)phosphoniumtetrafluorophopsphate), BrOP (bromotris(dimethylamino)phosphoniumhexafluorophosphate), DEPBT(3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) PyBrOP(bromotris(pyrrolidino)phosphonium hexafluorophosphate), EDC, HOAT,BOP—Cl and PyBrOP are preferred peptide coupling reagents. The amount ofpeptide coupling reagent is in the range of about 1.0 to about 10.0equivalents. Optional reagents that may be used in the amidebond-forming reaction include DMAP (4-dimethylaminopyridine) or activeester reagents, such as HURT (1-hydroxybenzotriazole), HOAT(hydroxyazabenzotriazole), HOSu (hydroxysuccinimide), HONB(endo-N-hydroxy-5-norbornene-2,3-dicarboxamide), in amounts ranging fromabout 1.0 to about 10.0 equivalents.

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine.

The term “alkyl” refers to saturated and unsaturated non-aromatichydrocarbon chains that may be a straight chain or branched chain,containing the indicated number of carbon atoms (these include withoutlimitation propyl, allyl, or propargyl), which may be optionallyinterrupted with N, O, or S. For example, C₁-C₁₀) indicates that thegroup may have from 1 to 10 (inclusive) carbon atoms in it. The term“alkylene” refers to a divalent alkyl (i.e., —R—).

The term “alkoxy” refers to an —O-alkyl radical.

The term “alkylenedioxo” refers to a divalent species of the structure—O—R—O—, in which R represents an alkylene.

The term “aminoalkyl” refers to an alkyl substituted with an aminogroup.

The term “mercapto” refers to an —SH radical.

The term “thioalkoxy” refers to an —S-alkyl radical.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may besubstituted by a substituent. Examples of aryl groups include phenyl andnaphthyl.

The terms “arylalkyl” and “aralkyl” refers to an alkyl substituted withan aryl.

The term “arylalkoxy” refers to an alkoxy substituted with an aryl.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, whereinthe cycloalkyl group additionally may be optionally substituted.Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, where the heteroatoms are selected from O, N,or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or Sif monocyclic, bicyclic, or tricyclic, respectively), and 0, 1, 2, 3, or4 atoms of each ring may be substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,and thiazolyl.

The terms “heteroarylalkyl” and “heteroaralkyl” refer to an alkylsubstituted with a heteroaryl.

The term “heteroarylalkoxy” refers to an alkoxy substituted with aheteroaryl.

The term “heterocyclyl” refers to a non-aromatic 5-8 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, where the heteroatoms areselected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,respectively), and 0, 1, 2 or 3 atoms of each ring may be substituted bya substituent. Examples of heterocyclyl groups include trizolyl,tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, andtetrahydrofuranyl.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further Substituted by one or more substituents.

The term “DMTr” refers to 4,4′-dimethoxytrityl, unless otherwisespecified.

The term “substituted” refers to the replacement of one or more hydrogenradicals in a given structure with the radical of a specifiedsubstituent including, but not limited to: halo, alkyl, alkenyl,alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl,arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl,alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl,arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino,trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl,arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl,alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl,carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl,heteroaryl, heterocyclic, and aliphatic. It is understood that thesubstituent can be further substituted.

The term “monosaccharide” embraces radicals of allose, altrose,arabinose, cladinose, erythrose, erythrulose, fructose, D-fucitol,L-fucitol, fucosamine, fucose, fuculose, galactosamine,D-galactosaminitol, N-acetyl-galactosamine, galactose, glucosamine,N-acetyl-glucosamine, glucosaminitol, glucose, glucose-6-phosphate,gulose glyceraldehyde. L-glycero-D-mannos-heptose, glycerol, glycerone,gulose, idose, lyxose, mannosamine, mannose, mannose-6-phosphate,psicose, quinovose, quinovosamine, rhamnitol, rhamnosamine, rhamnose,ribose, ribulose, sedoheptulose, sorbose, tagatose, talose, tartaricacid, threose, xylose and xylulose. The monosaccharide can be in D- orL-configuration. The monosaccharide may further be a deoxy sugar(alcoholic hydroxy group replaced by hydrogen), amino sugar (alcoholichydroxy group replaced by amino group), a thio sugar (alcoholic hydroxygroup replaced by thiol, or C═O replaced by C═S, or a ring oxygen ofcyclic form replaced by sulfur), a seleno sugar, a telluro sugar, an azasugar (ring carbon replaced by nitrogen), an imino sugar (ring oxygenreplaced by nitrogen), a phosphano sugar (ring oxygen replaced withphosphorus), a phospha sugar (ring carbon replaced with phosphorus), aC-substituted monosaccharide (hydrogen at a non-terminal carbon atomreplaced with carbon), an unsaturated monosaccharide, an alditol(carbonyl group replaced with CHOH group), aldonic acid (aldehydic groupreplaced by carboxy group), a ketoaldonic acid, a uronic acid, analdaric acid, and so forth. Amino sugars include amino monosaccharides,preferably galactosamine, glucosamine, mannosamine, fucosamine,quinovosamine, neuraminic acid, muramic acid, lactosediamine, acosamine,bacillosamine, daunosamine, desosamine, forosamine, garosamine,kanosamine, kansosamine, mycaminose, mycosamine, perosamine,pneumosamine, purpurosamine, rhodosamine. It is understood that themonosaccharide and the like can be further substituted.

The terms “disaccharide”, “trisaccharide” and “polysaccharide” embraceradicals of abequose, acrabose, amicetose, amylopectin, amylose, apiose,arcanose, ascarylose, ascorbic acid, boivinose, cellobiose, cellotriose,cellulose, chacotriose, chalcose, chitin, colitose, cyclodextrin,cymarose, dextrin, 2-deoxyribose, 2-deoxyglucose, diginose, digitalosedigitoxose, evalose, evemitrose, fructooligosaccharide,galto-oligosaccharide, gentianose, gentiobiose, glucan, glucogen,glycogen, hamamelose, heparin, inulin, isolevoglucosenone, isomaltose,isomaltotriose, isopanose, kojibiose, lactose, lactosamine,lactosediamine laminarabiose, levoglucosan, levoglucosenome, β-maltose,maltose, mannan-oligosaccharide, manninotriose, melezitose, melibiose,muramic acid, mycarose, mycinose, neuraminic acid, nigerose,nojirimycin, noviose, oleandrose, panose, paratose, planteose,primeverose, raffinose, rhodinose, rutinose, sarmentose, sedoheptulose,sedoheptulosan, solatriose, sophorose, stachyose, streptose, sucrose,α,α-trehalose, trehalosamine, turanose, tyvelose, xylobiose,umbelliferose and the like. Further, it is understood that the“disaccharide”, “trisaccharide” and “polysaccharide” and the like can befurther substituted. Disaccharide also includes amino sugars and theirderivatives, particularly, a mycaminose derivatized at the C-4′ positionor a 4 deoxy-3-amino-glucose derivatized at the C-6′ position.

Oligonucleotide

The oligonucleotide can be an siRNA, microRNA, antimicroRNA, microRNAmimics, antimiR, antagomir, dsRNA, ssRNA, aptamer, immune stimulatory,decoy oligonucleotides, splice altering oligonucleotides, triplexforming oligonucleotides, G-quadruplexes or antisense. In oneembodiment, the oligonucleotide is an IRNA agent.

In some embodiments, the oligonucleotide of the invention comprises oneor more monomers that are UNA (unlocked nucleic acid) nucleotides, UNArefers to an unlocked acyclic nucleic acid, wherein at least one of thebonds of the sugar has been removed, forming an unlocked “sugar”residue. In one example, UNA also encompasses monomer with bonds betweenC1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bondbetween the C1′ and C4′ carbons). In another example, the C2′-C3′ bond(i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons)of the sugar has been removed (see Fluiter et al., Mol. Biosyst., 2009,10, 1039, which is hereby incorporated by reference).

The term “iRNA agent” refers to an RNA agent (or can be cleaved into anRNA agent) which can down regulate the expression of a target gene(e.g., a siRNA), preferably an endogenous or pathogen target RNA. Whilenot wishing to be bound by theory, an iRNA agent may act by one or moreof a number of mechanisms, including post-transcriptional cleavage of atarget mRNA (referred to in the art as RNAi), or pre-transcriptional orpre-translational mechanisms. An iRNA agent can include a single strandor can include more than one strands, e.g., it can be a double strandediRNA agent. If the IRNA agent is a single strand it can include a 5′modification which includes one or more phosphate groups or one or moreanalogs of a phosphate group. In one preferred embodiment, the iRNAagent is double stranded.

The IRNA agent typically includes a region of sufficient homology to thetarget gene, and is of sufficient length in terms of nucleotides, suchthat the iRNA agent, or a fragment thereof, can mediate down regulationof the target gene. The iRNA agent is or includes a region which is atleast partially, and in some embodiments fully, complementary to thetarget RNA. It is not necessary that there be perfect complementaritybetween the iRNA agent and the target, but the correspondence ispreferably sufficient to enable the iRNA agent, or a cleavage productthereof, to direct sequence specific silencing, e.g., by RNAi cleavageof the target RNA, e.g., mRNA.

The nucleotides in the iRNA agent may be modified (e.g., one or morenucleotides may include a 2′-F or 2′-OCH₃ group), or be nucleotidesurrogates. The single stranded regions of an iRNA agent may be modifiedor include nucleoside surrogates, e.g., the unpaired region or regionsof a hairpin structure, e.g., a region which links two complementaryregions, can have modifications or nucleoside surrogates. Modificationto stabilize one or more 3′- or 5′-terminus of an iRNA agent, e.g.,against exonucleases. Modifications can include C3 (or C6, C7, C12)amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers(C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol),special biotin or fluorescein reagents that come as phosphoramidites andthat have another DMT-protected hydroxyl group, allowing multiplecouplings during RNA synthesis. Modifications can also include, e.g.,the use of modifications at the 2′ OH group of the ribose sugar, e.g.,the use of deoxyribonucleotides, e.g., deoxythymidine, instead ofribonucleotides, and modifications in the phosphate group, e.g.,phosphothioate modifications. In some embodiments, the different strandswill include different modifications.

In some embodiments, it is preferred that the strands be chosen suchthat the IRNA agent includes a single strand or unpaired region at oneor both ends of the molecule. A double stranded iRNA agent preferablyhas its strands paired with an overhang, e.g., one or two 5′ or 3′overhangs (preferably at least a 3′ overhang of 2-3 nucleotides).Preferred iRNA agents will have single-stranded overhangs, preferably 3′overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end.The overhangs can be the result of one strand being longer than theother, or the result of two strands of the same length being staggered.

Preferred lengths for the duplexed regions between the strands of theiRNA agent are between 6 and 30 nucleotides in length. The preferredduplexed regions are between 15 and 30, most preferably 18, 19, 20, 21,22, and 23 nucleotides in length. Other preferred duplexed regions arebetween 6 and 20 nucleotides, most preferably 6, 7, 8, 9, 10, 11 and 12nucleotides in length.

The oligonucleotide may be that described in U.S. Patent PublicationNos. 2009/0239814, 2012/0136042, 2013/0158824, or 2009/0247608, each ofwhich is hereby incorporated by reference.

A “single strand siRNA compound” as used herein, is an siRNA compoundwhich is made up of a single molecule. It may include a duplexed region,formed by intra-strand pairing, e.g., it may be, or include, a hairpinor pan-handle structure. Single strand siRNA compounds may be antisensewith regard to the target molecule

A single strand siRNA compound may be sufficiently long that it canenter the RISC and participate in RISC mediated cleavage of a targetmRNA. A single strand siRNA compound is at least 14, and in otherembodiments at least 15, 20, 25, 29, 35, 40, or 50 nucleotides inlength. In certain embodiments, it is less than 200, 100, or 60nucleotides in length.

Hairpin siRNA compounds will have a duplex region equal to or at least17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplexregion will may be equal to or less than 200, 100, or 50, in length. Incertain embodiments, ranges for the duplex region are 15-30, 17 to 23,19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin may havea single strand overhang or terminal unpaired region. In certainembodiments, the overhangs are 2-3 nucleotides in length. In someembodiments, the overhang is at the sense side of the hairpin and insome embodiments on the antisense side of the hairpin.

A “double stranded siRNA compound” as used herein, is an siRNA compoundwhich includes more than one, and in some cases two, strands in whichinterchain hybridization can form a region of duplex structure.

The antisense strand of a double stranded siRNA compound may be equal toor at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides inlength. It may be equal to or less than 200, 100, or 50, nucleotides inlength. Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides inlength. As used herein, term “antisense strand” means the strand of ansiRNA compound that is sufficiently complementary to a target molecule.e.g. a target RNA.

The sense strand of a double stranded siRNA compound may be equal to orat least 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length.It may be equal to or less than 200, 100, or 50, nucleotides in length.Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.

The double strand portion of a double stranded siRNA compound may beequal to or at least, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29,40, or 60 nucleotide pairs in length. It may be equal to or less than200, 100, or 50, nucleotides pairs in length. Ranges may be 15-30, 17 to23, 19 to 23, and 19 to 21 nucleotides pairs in length.

In many embodiments, the siRNA compound is sufficiently large that itcan be cleaved by an endogenous molecule, e.g., by Dicer, to producesmaller siRNA compounds, e.g., siRNAs agents

The sense and antisense strands may be chosen such that thedouble-stranded siRNA compound includes a single strand or unpairedregion at one or both ends of the molecule. Thus, a double-strandedsiRNA compound may contain sense and antisense strands, paired tocontain an overhang, e.g., one or two 5′ or 3′ overhangs, or a 3′overhang of 1-3 nucleotides. The overhangs can be the result of onestrand being longer than the other, or the result of two strands of thesame length being staggered. Some embodiments will have at least one 3′overhang. In one embodiment, both ends of an siRNA molecule will have a3′ overhang. In some embodiments, the overhang is 2 nucleotides.

In certain embodiments, the length for the duplexed region is between 15and 30, or 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe ssiRNA compound range discussed above. ssiRNA compounds can resemblein length and structure the natural Dicer processed products from longdsiRNAs. Embodiments in which the two strands of the ssiRNA compound arelinked, e.g., covalently linked are also included. Hairpin, or othersingle strand structures which provide the required double strandedregion, and a 3′ overhang are also contemplated.

The siRNA compounds described herein, including double-stranded siRNAcompounds and single-stranded siRNA compounds can mediate silencing of atarget RNA, e.g., mRNA, e.g., a transcript of a gene that encodes aprotein. For convenience, such mRNA is also referred to herein as mRNAto be silenced. Such a gene is also referred to as a target gene. Ingeneral, the RNA to be silenced is an endogenous gene or a pathogengene. In addition. RNAs other than mRNA, e.g., tRNAs, and viral RNAs,can also be targeted.

As used herein, the phrase “mediates RNAi” refers to the ability tosilence, in a sequence specific manner, a target RNA. While not wishingto be bound by theory, it is believed that silencing uses the RNAimachinery or process and a guide RNA, e.g., an ssiRNA compound of 21 to23 nucleotides.

In one embodiment, an siRNA compound is “sufficiently complementary” toa target RNA, e.g., a target mRNA, such that the siRNA compound silencesproduction of protein encoded by the target mRNA. In another embodiment,the siRNA compound is “exactly complementary” to a target RNA, e.g., thetarget RNA and the siRNA compound anneal, for example to form a hybridmade exclusively of Watson-Crick base pairs in the region of exactcomplementarity. A “sufficiently complementary” target RNA can includean internal region (e.g., of at least 10 nucleotides) that is exactlycomplementary to a target RNA. Moreover, in certain embodiments, thesiRNA compound specifically discriminates a single-nucleotidedifference. In this case, the siRNA compound only mediates RNAi if exactcomplementary is found in the region (e.g., within 7 nucleotides of) thesingle-nucleotide difference.

MicroRNAs

Micro RNAs (miRNAs) are a highly conserved class of small RNA moleculesthat are transcribed from DNA in the genomes of plants and animals, butare not translated into protein. Processed miRNAs are single stranded˜17-25 nucleotide (nt) RNA molecules that become incorporated into theRNA-induced silencing complex (RISC) and have been identified as keyregulators of development, cell proliferation, apoptosis anddifferentiation. They are believed to play a role in regulation of geneexpression by binding to the 3′-untranslated region of specific mRNAs.RISC mediates down-regulation of gene expression through translationalinhibition, transcript cleavage, or both. RISC is also implicated intranscriptional silencing in the nucleus of a wide range of eukaryotes.

The number of miRNA sequences identified to date is large and growing,illustrative examples of which can be found, for example, in: “miRBase:microRNA sequences, targets and gene nomenclature” Griffiths-Jones S,Grocock R J, van Dongen S, Bateman A, Enright A J, NAR, 2006, 34,Database Issue, D140-D144; “The microRNA Registry” Griffiths-Jones S.NAR, 2004, 32, Database Issue, D109-D111; and also athttp://microrna.sanger.ac.uk/sequences/.

Antisense Oligonucleotides

In one embodiment, a nucleic acid is an antisense oligonucleotidedirected to a target polynucleotide. The term “antisenseoligonucleotide” or simply “antisense” is meant to includeoligonucleotides that are complementary to a targeted polynucleotidesequence. Antisense oligonucleotides are single strands of DNA or RNAthat are complementary to a chosen sequence. e.g. a target gene mRNA.Antisense oligonucleotides are thought to inhibit gene expression bybinding to a complementary mRNA. Binding to the target mRNA can lead toinhibition of gene expression either by preventing translation ofcomplementary mRNA strands by binding to it, or by leading todegradation of the target mRNA. Antisense DNA can be used to target aspecific, complementary (coding or non-coding) RNA. If binding takesplaces this DNA/RNA hybrid can be degraded by the enzyme RNase H. Inparticular embodiments, antisense oligonucleotides contain from about 10to about 50 nucleotides, more preferably about 15 to about 30nucleotides. The term also encompasses antisense oligonucleotides thatmay not be exactly complementary to the desired target gene. Thus,instances where non-target specific-activities are found with antisense,or where an antisense sequence containing one or more mismatches withthe target sequence is the most preferred for a particular use, arecontemplated.

Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, can be usedto specifically inhibit protein synthesis by a targeted gene. Theefficacy of antisense oligonucleotides for inhibiting protein synthesisis well established. For example, the synthesis of polygalactauronaseand the muscarine type 2 acetylcholine receptor are inhibited byantisense oligonucleotides directed to their respective mRNA sequences(U.S. Pat. Nos. 5,739,119 and 5,759,829 each of which is incorporated byreference). Further, examples of antisense inhibition have beendemonstrated with the nuclear protein cyclin, the multiple drugresistance gene (MDG1). ICAM-1, E-selectins STK-1, striatal GABA_(A)receptor and human EGF (Jaskulski et al., Science, 1988 Jun. 10;240(4858):1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989;1(4):225-32; Peris et al., Brain Res Mol Brain Res. 1998 Jun. 15;57(2):310-20; U.S. Pat. Nos. 5,801,154; 5,789,573; 5,718,709 and5,610,288, each of which is incorporated by reference). Furthermore,antisense constructs have also been described that inhibit and can beused to treat a variety of abnormal cellular proliferations, e.g. cancer(U.S. Pat. Nos. 5,747,470; 5,591,317 and 5,783,683, each of which isincorporated by reference).

Methods of producing antisense oligonucleotides are known in the art andcan be readily adapted to produce an antisense oligonucleotide thattargets any polynucleotide sequence. Selection of antisenseoligonucleotide sequences specific for a given target sequence is basedupon analysis of the chosen target sequence and determination ofsecondary structure, T_(m), binding energy, and relative stability.Antisense oligonucleotides may be selected based upon their relativeinability to form dimers, hairpins, or other secondary structures thatwould reduce or prohibit specific binding to the target mRNA in a hostcell. Highly preferred target regions of the mRNA include those regionsat or near the AUG translation initiation codon and those sequences thatare substantially complementary to 5′ regions of the mRNA. Thesesecondary structure analyses and target site selection considerationscan be performed, for example, using v.4 of the OLIGO primer analysissoftware (Molecular Biology Insights) and/or the BLASTN 2.0.5 algorithmsoftware (Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

Antagomirs

Antagomirs are RNA-like oligonucleotides that harbor variousmodifications for RNAse protection and pharmacologic properties, such asenhanced tissue and cellular uptake. They differ from normal RNA by, forexample, complete 2′-O-methylation of sugar, phosphorothioate backboneand, for example, a cholesterol-moiety at 3′-end. Antagomirs may be usedto efficiently silence endogenous miRNAs by forming duplexes comprisingthe antagomir and endogenous miRNA, thereby preventing miRNA-inducedgene silencing. An example of antagomir-mediated miRNA silencing is thesilencing of miR-122, described in Krutzfeldt et al, Nature, 2005, 438:685-689, which is expressly incorporated by reference herein in itsentirety. Antagomir RNAs may be synthesized using standard solid phaseoligonucleotide synthesis protocols. See U.S. Patent ApplicationPublication Nos. 2007/0123482 and 2007/0213292 (each of which isincorporated herein by reference).

An antagomir can include ligand-conjugated monomer subunits and monomersfor oligonucleotide synthesis. Exemplary monomers are described in U.S.Patent Application Publication No. 2005/0107325, which is incorporatedby reference in its entirety. An antagomir can have a ZXY structure,such as is described in WO 2004/080406, which is incorporated byreference in its entirety. An antagomir can be complexed with anamphipathic moiety. Exemplary amphipathic moieties for use witholigonucleotide agents are described in WO 2004/080406, which isincorporated by reference in its entirety,

Aptamers

Aptamers are nucleic acid or peptide molecules that bind to a particularmolecule of interest with high affinity and specificity (Tuerk and Gold,Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990),each of which is incorporated by reference in its entirely). DNA or RNAaptamers have been successfully produced which bind many differententities from large proteins to small organic molecules. See Eaton.Curr. Opin. Chem. Biol. 1:10-16 (1997), Famulok, Curr. Opin. Struct.Biol. 9:324-9(1999), and Hermann and Patel, Science 287:820-5 (2000),each of which is incorporated by reference in its entirety. Aptamers maybe RNA or DNA based, and may include a riboswitch. A riboswitch is apart of an mRNA molecule that can directly bind a small target molecule,and whose binding of the target affects the gene's activity. Thus, anmRNA that contains a riboswitch is directly involved in regulating itsown activity, depending on the presence or absence of its targetmolecule. Generally, aptamers are engineered through repeated rounds ofin vitro selection or equivalently, SELEX (systematic evolution ofligands by exponential enrichment) to bind to various molecular targetssuch as small molecules, proteins, nucleic acids, and even cells,tissues and organisms. The aptamer may be prepared by any known method,including synthetic, recombinant, and purification methods, and may beused alone or in combination with other aptamers specific for the sametarget. Further, as described more fully herein, the term “aptamer”specifically includes “secondary aptamers” containing a consensussequence derived from comparing two or more known aptamers to a giventarget.

Ribozymes

According to another embodiment, nucleic acid-lipid particles areassociated with ribozymes. Ribozymes are RNA molecules complexes havingspecific catalytic domains that possess endonuclease activity (Kim andCech, Proc Natl Acad Sci USA. 1987 December; 84(24):8788-92; Forster andSymons, Cell. 1987 Apr. 24:49(2):211-20). For example, a large number ofribozymes accelerate phosphoester transfer reactions with a high degreeof specificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., Cell. 1981 Dec.; 27(3 Pt2):487-96; Michel and Westhof, J Mol Biol. 1990 Dec. 5; 216(3):585-610;Reinhold-Hurek and Shub, Nature. 1992 May 14; 357(6374):173-6). Thisspecificity has been attributed to the requirement that the substratebind via specific base-pairing interactions to the internal guidesequence (“IGS”) of the ribozyme prior to chemical reaction.

At least six basic varieties of naturally-occurring enzymatic RNAs areknown presently. Each can catalyze the hydrolysis of RNA phosphodiesterbonds in trans (and thus can cleave other RNA molecules) underphysiological conditions. In general, enzymatic nucleic acids act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of a enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif, forexample. Specific examples of hammerhead motifs are described by Rossiel al. Nucleic Acids Res. 1992 Sep. 11; 20(17):4559-65. Examples ofhairpin motifs are described by Hampel et al. (Eur. Pat. Appl. Publ. No.EP 0360257), Hampel and Tritz, Biochemistry 1989 Jun. 13;28(12):4929-33; Hampel et al., Nucleic Acids Res. 1990 Jan.25:18(2):299-304 and U.S. Pat. No. 5,631,359. An example of thehepatitis δ virus motif is described by Perrotta and Been, Biochemistry.1992 Dec. 1; 31(47):11843-52; an example of the RNaseP motif isdescribed by Guerrier-Takada et al., Cell. 1983 Dec.; 35(3 Pt 2):849-57;Neurospora VS RNA ribozyme motif is described by Collins (Saville andCollins, Cell. 1990 May 18; 61(4):685-96; Saville and Collins, Proc NatlAcad Sci USA. 1991 Oct. 1; 88(19):8826-30; Collins and Olive,Biochemistry. 1993 Mar. 23:32(11):2795-9); and an example of the Group Iintron is described in U.S. Pat. No. 4,987,071. Importantcharacteristics of enzymatic nucleic acid molecules used are that theyhave a specific substrate binding site which is complementary to one ormore of the target gene DNA or RNA regions, and that they havenucleotide sequences within or surrounding that substrate binding sitewhich impart an RNA cleaving activity to the molecule. Thus the ribozymeconstructs need not be limited to specific motifs mentioned herein.

Methods of producing a ribozyme targeted to any polynucleotide sequenceare known in the art. Ribozymes may be designed as described in Int.Pat. Appl. Publ. Nos. WO 93/23569 and WO 94/02595, each specificallyincorporated herein by reference, and synthesized to be tested in vitroand in viva, as described therein.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. Nos. WO 92/07065. WO 93/15187, and WO91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; U.S. Pat. No. 5,334,711;and Int. Pat. Appl. Publ. No. WO 94/13688, which describe variouschemical modifications that can be made to the sugar moieties ofenzymatic RNA molecules), modifications which enhance their efficacy incells, and removal of stem II bases to shorten RNA synthesis times andreduce chemical requirements.

Immunostimulatory Oligonucleotides

Nucleic acids associated with lipid particles may be immunostimulatory,including immunostimulatory oligonucleotides (ISS; single- ordouble-stranded) capable of inducing an immune response whenadministered to a subject, which may be a mammal or other patient. ISSinclude, e.g., certain palindromes leading to hairpin secondarystructures (see Yamamoto S., et al. (1992) J. Immunol. 148: 4072-4076,which is incorporated by reference in its entirety), or CpG motifs, aswell as other known ISS features (such as multi-G domains, see WO96/11266, which is incorporated by reference in its entirety).

The immune response may be an innate or an adaptive immune response. Theimmune system is divided into a more innate immune system, and acquiredadaptive immune system of vertebrates, the latter of which is furtherdivided into humoral cellular components. In particular embodiments, theimmune response may be mucosal.

In particular embodiments, an immunostimulatory nucleic acid is onlyimmunostimulatory when administered in combination with a lipidparticle, and is not immunostimulatory when administered in its “freeform.” Such an oligonucleotide is considered to be immunostimulatory.

Immunostimulatory nucleic acids are considered to be non-sequencespecific when it is not required that they specifically bind to andreduce the expression of a target polynucleotide in order to provoke animmune response. Thus, certain immunostimulatory nucleic acids maycomprise a sequence corresponding to a region of a naturally occurringgene or mRNA, but they may still be considered non-sequence specificimmunostimulatory nucleic acids.

In one embodiment, the immunostimulatory nucleic acid or oligonucleotidecomprises at least one CpG dinucleotide. The oligonucleotide or CpGdinucleotide may be unmethylated or methylated. In another embodiment,the immunostimulatory nucleic acid comprises at least one CpGdinucleotide having a methylated cytosine. In one embodiment, thenucleic acid comprises a single CpG dinucleotide, wherein the cytosinein said CpG dinucleotide is methylated. In an alternative embodiment,the nucleic acid comprises at least two CpG dinucleotides, wherein atleast one cytosine in the CpG dinucleotides is methylated. In a furtherembodiment, each cytosine in the CpG dinucleotides present in thesequence is methylated. In another embodiment, the nucleic acidcomprises a plurality of CpG dinucleotides, wherein at least one of saidCpG dinucleotides comprises a methylated cytosine.

Linker

The Linker can be any suitable group for coupling the oligonucleotide tothe Ligands). Other examples of Linkers are described in InternationalPublication No. WO 2009/082607 and U.S. Patent Publication Nos.2009/0239814, 2012/0136042, 2013/0158824, or 2009/0247608, each of whichis hereby incorporated by reference.

Attachment Point of Oligonucleotide to Linker

The oligonucleotide can be attached to the Linker via any suitable groupfor coupling the two. The group can be cleavable or non-cleavable.Examples or Linkers and suitable coupling groups are described herein.Other examples of coupling groups are described in InternationalPublication No. WO 2009/082607 and U.S. Patent Publication Nos.2009/0239814, 2012/0136042, 2013/0158824, or 2009/0247608, each of whichis hereby incorporated by reference. Suitable coupling groups include,for example, NR⁸, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, suchas, but not limited to, alkyl, alkenyl, alkynyl, arylalkyl, arylalkenyl,arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, each of which may be substituted or unsubstituted,and which one or more methylenes can be interrupted or terminated by O,S, S(O), SO₂, N(R⁸), C(O), substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, or substituted or unsubstitutedheterocyclic, where R⁸ is hydrogen, acyl, aliphatic or substitutedaliphatic.

A cleavable group is one which is sufficiently stable outside the cell,but which upon entry into a target cell is cleaved to release the twoparts the group is holding together. In a preferred embodiment, thecleavable group is cleaved at least 10 times or more, preferably atleast 100 times faster in the target cell or under a first referencecondition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum).

Cleavable groups are susceptible to cleavage agents, e.g., pH, redoxpotential or the presence of degradative molecules. Generally, cleavageagents are more prevalent or found at higher levels or activities insidecells than in serum or blood. Examples of such degradative agentsinclude: redox agents which are selected for particular substrates orwhich have no substrate specificity, including, e.g., oxidative orreductive enzymes or reductive agents such as mercaptans, present incells, that can degrade a redox cleavable group by reduction: esterases;endosomes or agents that can create an acidic environment, e.g., thosethat result in a pH of five or lower; enzymes that can hydrolyze ordegrade an acid cleavable group by acting as a general acid, peptidases(which can be substrate specific), and phosphatases.

A cleavable group, such as a disulfide bond can be susceptible to pH.The pH of human serum is 7.4, while the average intracellular pH isslightly lower, ranging from about 7.1-7.3. Endosomes have a more acidicpH, in the range of 5.5-6.0, and lysosomes have an even more acidic pHat around 5.0. Some linkers will have a cleavable group that is cleavedat a preferred pH, thereby releasing the cationic lipid from the ligandinside the cell, or into the desired compartment of the cell.

A conjugate can include a cleavable group that is cleavable by aparticular enzyme. The type of cleavable group incorporated into aconjugate can depend on the cell to be targeted. For example, livertargeting ligands can be linked to the cationic lipids through achemical moiety that includes an ester group. Liver cells are rich inesterases, and therefore the group will be cleaved more efficiently inliver cells than in cell types that are not esterase-rich. Othercell-types rich in esterases include cells of the lung, renal cortex,and testis.

Coupling groups that contain peptide bonds can be used when targetingcell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable group can beevaluated by testing the ability of a degradative agent (or condition)to cleave the candidate group. It will also be desirable to also testthe candidate cleavable group for the ability to resist cleavage in theblood or when in contact with other non-target tissue. Thus one candetermine the relative susceptibility to cleavage between a first and asecond condition, where the first is selected to be indicative ofcleavage in a target cell and the second is selected to be indicative ofcleavage in other tissues or biological fluids, e.g., blood or serum.The evaluations can be carried out in cell free systems, in cells, incell culture, in organ or tissue culture, or in whole animals. It may beuseful to make initial evaluations in cell-free or culture conditionsand to confirm by further evaluations in whole animals. In preferredembodiments, useful candidate compounds are cleaved at least 2, 4, 10 or100 times faster in the cell (or under in vitro conditions selected tomimic intracellular conditions) as compared to blood or serum (or underin vitro conditions selected to mimic extracellular conditions).

i. Redox Cleavable Groups

One class of cleavable groups are redox cleavable groups that arecleaved upon reduction or oxidation. An example of reductively cleavablegroup is a disulphide linking group (—S—S—). To determine if a candidatecleavable group is a suitable “reductively cleavable linking group.” orfor example is suitable for use with a particular iRNA moiety andparticular targeting agent one can look to methods described herein. Forexample, a candidate can be evaluated by incubation with dithiothreitol(DTT), or other reducing agent using reagents know in the art, whichmimic the rate of cleavage which would be observed in a cell, e.g., atarget cell. The candidates can also be evaluated under conditions whichare selected to mimic blood or serum, conditions. In a preferredembodiment, candidate compounds are cleaved by at most 10% in the blood.In preferred embodiments, useful candidate compounds are degraded atleast 2, 4, 10 or 100 times faster in the cell (or under in vitroconditions selected to mimic intracellular conditions) as compared toblood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

ii. Phosphate-Based Cleavable Groups

Phosphate-based cleavable groups are cleaved by agents that degrade orhydrolyze the phosphate group. An example of an agent that cleavesphosphate groups in cells are enzymes such as phosphatases in cells.Examples of phosphate-based linking groups are —O—P(O)(OR^(k))—O—,—O—P(S)(OR^(k))—O—, —O—P(S)(SR^(k))—O—, —S—P(O)(OR^(k))—O—,—O—P(O)(OR^(k))—S—, —S—P(O)(OR^(k))—S—, —O—P(S)(OR^(k))—S—,—S—P(S)(OR^(k))—O—, —O—P(O)(R^(k))—O—, —O—P(S)(R^(k))—O—,—S—P(O)(R^(k))—O—, —S—P(S)(R^(k))—O—, —S—P(O)(R^(k))—S—,—O—P(S)(R^(k))—S—. Preferred embodiments are —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, and—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

iii. Acid Cleavable Groups

Acid cleavable groups are linking groups that are cleaved under acidicconditions. In preferred embodiments acid cleavable groups are cleavedin an acidic environment with a pH of about 6.5 or lower (e.g., about6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as ageneral acid. In a cell, specific low pH organelles, such as endosomesand lysosomes can provide a cleaving environment for acid cleavablelinking groups. Examples of acid cleavable groups include but are notlimited to hydrazones, esters, and esters of amino acids. Acid cleavablegroups can have the general formula —C═NN—, C(O)O, or —OC(O). Apreferred embodiment is when the carbon attached to the oxygen of theester (the alkoxy group) is an aryl group, substituted alkyl group, ortertiary alkyl group such as dimethyl pentyl or t-butyl. Thesecandidates can be evaluated using methods analogous to those describedabove.

iv. Ester-Based Groups

Ester-based cleavable groups are cleaved by enzymes such as esterasesand amidases in cells. Examples of ester-based cleavable groups includebut are not limited to esters of alkylene, alkenylene and alkynylenegroups. Ester cleavable linking groups have the general formula —C(O)O—,or —OC(O)—. These candidates can be evaluated using methods analogous tothose described above.

v. Peptide-Based Cleaving Groups

Peptide-based cleavable groups are cleaved by enzymes such as peptidasesand proteases in cells. Peptide-based cleavable groups are peptide bondsformed between amino acids to yield oligopeptides (e.g., dipeptides,tripeptides etc.) and polypeptides. Peptide-based cleavable groups donot include the amide group (—C(O)NH—). The amide group can be formedbetween any alkylene, alkenylene or alkynelene. A peptide bond is aspecial type of amide bond formed between amino acids to yield peptidesand proteins. The peptide based cleavage group is generally limited tothe peptide bond (i.e., the amide bond) formed between amino acidsyielding peptides and proteins and does not include the entire amidefunctional group. Peptide-based cleavable linking groups have thegeneral formula —NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) arethe R groups of the two adjacent amino acids. These candidates can beevaluated using methods analogous to those described above. As usedherein. “carbohydrate” refers to a compound which is either acarbohydrate per sc made up of one or more monosaccharide units havingat least 6 carbon atoms (which may be linear, branched or cyclic) withan oxygen, nitrogen or sulfur atom bonded to each carbon atom; or acompound having as a part thereof a carbohydrate moiety made up of oneor more monosaccharide units each having at least six carbon atoms(which may be linear, branched or cyclic), with an oxygen, nitrogen orsulfur atom bonded to each carbon atom. Representative carbohydratesinclude the sugars (mono-, di-, tri- and oligosaccharides containingfrom about 4-9 monosaccharide units), and polysaccharides such asstarches, glycogen, cellulose and polysaccharide gums. Specificmonosaccharides include C5 and above (preferably C₅-C₈) sugars; di- andtrisaccharides include sugars having two or three monosaccharide units(preferably C₅-C₈),

The Ligands

The Ligand can be any ligand described herein.

Other suitable ligands are described in U.S. Patent Publication Nos.2009/0239814, 2012/0136042, 2013/0158824, or 2009/0247608, each of whichis hereby incorporated by reference.

Formulations

The conjugates described herein can be formulated fir administration toa subject. For ease of exposition the formulations, compositions andmethods in this section are discussed largely with regard to conjugatesof unmodified iRNA agents. It will understood, however, that theseformulations, compositions and methods can be practiced with conjugatesof other oligonucleotides, e.g., modified iRNA agents, and such practiceis within the invention.

A formulated iRNA conjugate can assume a variety of states. In someexamples, the conjugate is at least partially crystalline, uniformlycrystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10%water). In another example, the iRNA conjugate is in an aqueous phase.e.g., in a solution that includes water.

The aqueous phase or the crystalline conjugates can, e.g., beincorporated into a delivery vehicle, e.g., a liposome (particularly forthe aqueous phase) or a particle (e.g., a microparticle as can beappropriate for a crystalline composition). Generally, the iRNAconjugate is formulated in a manner that is compatible with the intendedmethod of administration. The iRNA conjugate can be incorporated into anucleic acid lipid nanoparticle. In one embodiment, each nanoparticleincludes the conjugate, a cationic lipid (e.g., a cationic lipid havinga pK_(a) ranging from about 4 to about 11, such as from about 5 to about7), a non-cationic lipid (such as a neutral lipid), an aggregationreducing agents (such as polyethylene glycol (PEG) or PEG-modifiedlipid), and optionally a sterol (e.g., cholesterol).

In particular embodiments, the composition is prepared by at least oneof the following methods: spray drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques; orsonication with a lipid, freeze-drying, condensation and otherself-assembly.

An iRNA conjugate can be formulated in combination with another agent,e.g., another therapeutic agent or an agent that stabilizes an iRNA,e.g., a protein that complexes with iRNA to form an iRNP. Still otheragents include chelators, e.g., EDTA (e.g., to remove divalent cationssuch as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAseinhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA composition includes at least one secondtherapeutic agent (e.g., an agent other than an RNA or a DNA). Forexample, an iRNA composition for the treatment of a viral disease, e.g.,HIV, might include a known antiviral agent (e.g., a protease inhibitoror reverse transcriptase inhibitor). In another example, an iRNAcomposition for the treatment of a cancer might further comprise achemotherapeutic agent.

The iRNA conjugate may be formulated with a drug that affects (forexample, increases) the uptake of the iRNA agent into a cell. The drugcan be administered before, after, or at the same time that the iRNAagent is administered. The drug can also be covalently linked to theiRNA agent. The drug can be, for example a lipopolysaccharide, anactivator of p38 MAP kinase, or an activator of NF-κB. The drug can havea transient effect on the cell.

In one embodiment, the drug used increases the uptake of the iRNA agentinto the cell, for example, by disrupting the cell's cytoskeleton e.g.,by disrupting the cell's microtubules, microfilaments, and/orintermediate filaments. The drug can be, for example, taxon,vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide,latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

The drug can also increase the uptake of the IRNA conjugate into thecell, for example, by activating an inflammatory response. Exemplarydrug's that would have such an effect include tumor necrosis factoralpha (TNF-α), interleukin-1 beta, or gamma interferon.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference.

EXAMPLES

Abbreviations: TBAHS is Tetrabutylammonium hydrogen sulfate; DCM isdichloromethane; NHS is N-hydroxysuccinamide; DIEA isN,N-diisopropylethylamine; EDC is1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide.

Example 1: Synthesis of Triantennary GalNAc Monomers with AcyclicLinker-1

The triantennary GalNAc moieties 211a-d are synthesized as shown inScheme 1 below,

i) NaBH₄, MeOH; ii) CH₂Cl₂, MsCl, NEt₃; iii) T or N4-Bz-C or N6-A orN2-iBu-G, CsCO₃, NaI, DMF, 110° C., 20-60 min; iv) a) DSC, NEt₃, b)N6-Phth-hexanediamine, CH₂Cl₂; v AcOH; vi) DMTrCl, py; vii) MeNH₂; viii)R1 or R2, EDCI, DIPEA, CH₂Cl₂; ix) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, DIPEA. CH₂Cl₂

i. Synthesis of(S)-1-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)ethane-1,2-diol 201

To a stirred solution of the commercially available (R)-methyl2-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-2-hydroxyacetate 200 (1.9 g) inmethanol (60 mL), solid sodium borohydride (0.4 g) is added at 0-5° C.,and after the addition the reaction mixture is warmed to roomtemperature and stirred for 20 min. The reaction mixture is diluted withsaturated ammonium chloride and extracted with dichloromethane. Theorganic layer is dried over Na₂SO₄ and concentrated to isolate the crudeproduct (1.6 g) which is used as such in the next step without furtherpurification.

ii. Synthesis of(S)-2-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-2-hydroxyethylmethanesulfonate 202

To a solution of the alcohol 201 (1.6 g) in dichloromethane (60 mL)triethylamine (1.5 mL) is added and the mixture cooled with stirring. Tothis stirred solution methanesulfonyl chloride (1.3 g) indichloromethane (30 mL) is added drop wise and the mixture is stirred atroom temperature for two hours after which the reaction mixture iswashed with satd. NaHCO₃ solution (100 mL) followed by brine (100 mL)and the organic layer after drying over anhyd. Na₂SO₄ is concentrated.The thus obtained crude product on purification using silica gelprovided the pure product.

iii. General Procedure for the Synthesis of 203a-d

In a general procedure the nucleo base (2 eq.) is dissolved in anhydrousDMF (100 mL) at 110° C. and to this stirred solution solid CsCO₃ (2 g)and sodium iodide (2 g) is added and the mixture stirred vigorously. Tothis stirred solution a solution of the mesylate 202 (1 eq.) inanhydrous DMF (10 mL) is added dropwise. The reaction mixture afterstirring at 110° C. for an additional 30 min. is concentrated underreduced pressure. The residue is dissolved in ethyl acetate and theorganic layer is washed with satd. NaHCO₃ (100 mL), brine (100 mL) anddried (anhyd. Na₂SO₄). The concentration of the organic layer providedthe crude product which is purified by flash column chromatography toisolate the pure product 203a-d.

iv. Synthesis of 204a-d

The alcohol 203a-d is initially treated with disuccinimidylcarbonate(DSC) in the presence of triethylamine to afford the succinimidylesterwhich on treatment with monophthalimidoprotected hexanediamine in thepresence of pyridine provide the amine substituted products 204a-d aftercolumn purification.

v. Synthesis of 205a-d

The acetonide protection in 204a-d is removed by treating with aceticacid under reported conditions to afford 205a-d which is used in thenext step as such.

vi. Synthesis of 206a-d

Treatment of the dial 205a-d with DMTrCl in pyridine would provide themono DMT protected alcohol 206a-d.

vii. Synthesis of 207a-d

The phthalimido protected amines 206a-d are treated with a solution ofmethylamine in methanol (20×) at room temperature overnight. Theconcentration of the reaction mixture followed by column purificationprovides the deprotected amines 207a-d.

viii. Synthesis of 208a-d

Coupling of the monoantennary GalNAc containing carboxylic acid with theamines 207a-d using EDC and Hunig's base would lead to the ligandconjugated monomers 208a-d.

ix. Synthesis of 209a-d

Using a similar coupling procedure coupling of the triantennary GalNAccontaining carboxylic acid with the amines 207a-d would lead to thetriantennary GalNAc conjugated monomers 209a-d

x. Synthesis of 210a-d and 211a-d

Phosphitylation of the alcohols 208a-d and 209a-d using 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite in the presence of Hunig's base inCH₂Cl₂ provided the corresponding amidites 210a-d and 211a-d after flashcolumn purification.

Example 2: Synthesis of Triantennary GalNAc Monomers with AcyclicLinker-2

The triantennary GalNAc moieties 219 are synthesized as shown in Scheme2 below.

Example 3: Synthesis of Triantennary GalNAc Monomers with AcyclicLinker-3

The triantennary GalNAc moiety 215 is synthesized as shown in Scheme 3below.

i. Synthesis of 222

The commercially available (R)-glycidol was converted to the ODMTrprotected epoxide 220 as reported in the literature. Treatment of theepoxide 220 (1.12 g, 3 mmol) with the 1,6-dimethylaminohexane 221 (6mmol, 2 eq.) at 110° C., under microwave irradiation for 30 min providedthe epoxide opened product 222 in 90% yield.

ii. Synthesis of 224

The coupling of the amine 222 with the carboxylic acid 223 provided thecoupled product 224 in good yields.

iii. Synthesis of 225

Phosphitylation of the alcohols 224 using 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite in the presence of Hunig's base inCH₂Cl₂ provided the corresponding amidite 225 after flash columnpurification.

Example 4: General Structure for Tri-Antennary α-Anomer—ConjugateBuilding Block

The tri-antennary α-anomers having the formula below are prepared.

For example, R, R′, and R″ may be independently, C₆-C₁₀ aryl, C₆-C₁₀heteroaromatic, C₁-C₂₀ alkyl, or a sugar (e.g., galactose or GalNAc).

The tri-antennary α-anomers having the formula below are also prepared.

-   -   X′—NH—C(O)—, —C(O)NH—, —O—C(O)—NH—, —NH—C(O)—O—, —NH—C(O)—NH—,        —O—    -   =alkylene, polyethylene glycol

For example, R, R′, and R″ may, be independently, C₆-C₁₀ aryl, C₆-C₁₀heteroaromatic, C₁-C₂₀ alkyl, or a sugar (e.g., galactose or GalNAc).

Example 5: General Structure of Tri-antennary β-anomer-ConjugateBuilding Block

The tri-antennary β-anomers having the formula below are prepared.

For example, R, R′, and R″ may be independently, C₆-C₁₀ aryl, C₆-C₁₀heteroaromatic, C₁-C₂₀ alkyl, or a sugar (e.g., galactose or GalNAc).

Example 6: General Structure of Bi-Antennary α-Anomer-Conjugate BuildingBlock

The bi-antennary α-anomers having the formula below are prepared.

For example, R, R′, and R″ may be independently, C₆-C₁₀ aryl, C₆-C₁₀heteroaromatic, C₁-C₂₀ alkyl, or a sugar (e.g., galactose or GalNAc).

Example 7: Synthesis Mono-, Bi- and Tri-Conjugate Building Blocks

The mono-, bi- and tri-conjugate building blocks may be prepared fromaide intermediates as shown below,

Example 8: Synthesis of Intermediates of ASGPR Ligand (Schemes 4-8)

Intermediates useful for preparing ASGPR ligands may be synthesized asshown in Schemes 4-8 below.

Synthesis of compound 9: N-Acetyl glucosamine (50 g, 226 mmol) are takenin allyl alcohol and is heated at 90° C. for 24 hr. The reaction mixtureis cooled to room temperature and the ally alcohol is removed bydistillation. The residue is dissolved in pyridine and reacted withpivaloyl chloride to obtain the compound 3. The pivaloyl ester isreacted with triflic anhydride in pyridine for 2 hrs and the mixture isquenched by adding water. The mixture is heated at 90° C. for 24 h toobtain the product 4. The ester groups are removed by treatment withsodium methoxide and compound 5 isolated. The hydroxyl groups areprotected by dimethoxy propane to obtain the product 6. It is thenreacted with tosyl chloride and sodium azide in DMF to obtain thecompound 7. Compound 7 is subjected to Click cyclo-addition andepoxidation with MCPBA to generate the compound 9.

Synthesis of compound 13: The epoxide 13 is reacted with ammonia in thepresence of LiClO₄ to generate the amino alcohol 10. This is coupledwith N-Cbz amino hexanoic acid under peptide coupling conditions toobtain the compound 11. Removal of acetonide protection and benzoylationof the hydroxyl groups generate the compound 12. Hydrogenation ofcompound 12 using Pd/C in WON generates the amine 13 as a TFA salt.

Synthesis of compound 17: Under peptide coupling conditions, the amine13 is reacted with tricarboxylic acid 14 to generate the compound 15.Hydrogenation yields the compound 16, which is then reacted withmonobenzyl dodecane dioic acid to provide the protected tri-antennaryintermediate 17.

Synthesis of hydroxy proline intermediate 20: Compound 17 ishydrogenated under balloon pressure to obtain the carboxylic acid 18,which is then reacted with amine 19 to generate compound 20 underpeptide coupling conditions.

Synthesis of solid support 21: The hydroxy proline 20 is treated withsuccinic anhydride and DMAP to generate a succinate derivative. Thissuccinate derivative is loaded onto a solid support using peptidecoupling conditions to obtain the support 21.

Example 9: Synthesis of Tri-Antennary β-Anomer—Conjugate Building Block(Schemes 9-10)

Synthesis of carboxylic acid 29: Compound 22 is reacted with the alcohol23 in the presence of TMSOTf in DCE to generate the carboxylate 24.Acetate groups are removed by TEA in MeOH to obtain the compound 25.DMTr is introduced in the O-6 position and the hydroxyl groups arebenzoylated to obtain compound 26. Removal of DMTr group under acidconditions and reaction with Ms-Cl generate the mesyl derivative.Reaction of this mesyl derivative with sodium azide give the C-6 azidoderivative 27. Click reaction and the deprotection of the methyl estergive the carboxylic acid 29.

Synthesis of solid support 32: The carboxylic acid 29 is reacted withTri-antennary amine under peptide coupling conditions generate thetri-antennary derivative 31. This is hydrogenated and the carboxylicacid is reacted with hydroxy proline. This intermediate is reacted withsuccinic anhydride and the succinate is loaded on to the solid supportto get compound 32.

Example 10: Synthesis of Bi-Antennary α-Anomer—Conjugate Building Block(Schemes 11-14)

Synthesis of carboxylic acid 36: Hydroxy proline derivative 33 isreacted with monobenzyl hexane dioic acid using HBTU/DIEA to generatethe carboxylate 35, which is further hydrogenated to yield thecarboxylic acid 36.

Synthesis of Bi-antennary solid support 41: N-Boc glutamic acid isreacted with the amine 13 using HBTU/DIEA give compound 38. Deprotectionof the Boc protecting group and reaction of the carboxylic acid 36 withthis amine yield the hydroxy proline derivative 39. Removal of TBDMS andreaction of succinic anhydride with this hydroxyl group generate thesuccinate derivative. This is loaded onto the solid support to give thehi-antennary solid support 41.

Similarly, compound 54 can be prepared as shown in Schemes 13 and 14below.

Example 11: Synthesis of Triantennary α-Anomeric Building Block

The triantennary α-anomeric building block 55 can be prepared by scheme15 below.

Example 12: Synthesis of Tri-Antennary α-Anomer siRNA Conjugate

Using the conjugate building block 21 described earlier (see scheme 8),RNA is synthesized with the ligand attached to 3′-end of the sensestrand according to known procedures. This is annealed with an antisensestrand. The product is shown below.

Example 13: Synthesis of Tri-Antennary β-Anomer siRNA Conjugate

Using the conjugate building block 32 described earlier (see scheme 10),RNA is synthesized with the ligand attached to 3′-end of the sensestrand according to known procedures. This is annealed with an antisensestrand. The product is shown below.

Example 14: Synthesis of Bi-Antennary α-Anomer siRNA Conjugate

Using the conjugate building block 41 described earlier (see scheme 12),RNA is synthesized with the ligand attached to 3′-end of the sensestrand according to known procedures. This is annealed with an antisensestrand.

Example 15: Synthesis of Mono-GalNAc Building Blocks for OligonucleotideConjugation

The Mono-GalNAc building blocks 104 and 105 are prepared as shown inScheme 16.

Synthesis of 102: GalNAc acid 100 (8.39 g, 18.71 mmol) and hydroxyproline amine (10.00 g, 18.77 mmol) were taken together indichloromethane. HBTU (10.68 g, 28.12 mmol) and DIEA (9.80 mL, 3 eq.)were added and stirred the mixture for 2 hrs at ambient temperature. Theproduct was TLC checked and the reaction mixture was transferred to aseparatory funnel and washed with water and brine. The organic layer wasdried over sodium sulfate and the solvent was removed. The crude productwas purified by silica gel chromatography using dichloromethane and MeOHas solvents to get the compound 102 as a pale yellow fluffy solid (11.77g, 63%). ¹H NMR (400 MHz, DMSO) δ 7.80 (d, J=9.2 Hz, 1H), 7.69 (t, J=5.6Hz, 1H), 7.39-7.09 (m, 9H), 6.86 (ddd, J=9.0, 5.4, 2.1 Hz, 4H), 5.20 (d,J=3.4 Hz, 1H), 5.03-4.83 (m, 2H), 4.47 (d, J=8.5 Hz, 1H), 4.41-4.07 (m,2H), 4.04-3.95 (m, 3H), 3.86 (dt, J=11.2, 8.9 Hz, 1H), 3.79-3.68 (m,6H), 3.68-3.36 (m, 3H), 3.21-2.88 (m, 5H), 2.26-2.14 (m, 2H), 2.09 (s,3H), 2.02 (t, J=6.7 Hz, 2H), 1.98 (s, 3H), 1.87 (d, J=7.5 Hz, 3H), 1.76(s, 3H), 1.53-1.29 (m, 7H).

Synthesis of 104: Hydroxy proline derivative 102 (6.00 g, 6.24 mmol) wasdissolved in dichloromethane (100 mL). DIEA (2.20 mL, 3 eq) andchloroamidite reagent were added, the reaction mixture was stirred for30 minutes and TLC checked. It was transferred to a separatory funneland washed with water and sodium bicarbonate solution. The organic layerwas dried over sodium sulfate and the crude product was purified bysilica gel chromatography using dichloromethane and WON as eluent to getthe compound as a white fluffy solid. ¹H NMR (400 MHz, DMSO), δ 7.80 (d,J=9.2 Hz, 1H), 7.68 (s, 1H), 7.42-7.06 (m, 8H), 7.01-6.73 (m, 4H), 5.20(d, J=3.3 Hz, 1H), 4.96 (dd, J=11.2, 3.3 Hz, 1H), 4.63 (d, J=4.7 Hz,1H), 4.47 (d, J=8.5 Hz, 1H), 4.15 (s, 1H), 4.01 (s, 3H), 3.86 (d, J=11.0Hz, 1H), 3.70 (d, J=16.5 Hz, 9H), 3.45 (ddd, J=37.0, 23.3, 16.4 Hz, 6H),2.99 (dd, J=12.3, 6.4 Hz, 3H), 2.74 (dd, J=9.2, 5.8 Hz, 2H), 2.21 (s,2H), 2.09 (s, 3H), 2.05-1.95 (m, 5H), 1.88 (s, 3H), 1.76 (s, 3H),1.52-1.16 (m, 11H), 1.16-1.02 (m, 11H). ³¹P NMR δ=151.78, 151.61,151.50, 151.30.

Synthesis of 105: Compound 102 (2.10 g, 2.18 mmol) was dissolved in DCM(20 mL). To this mixture succinic anhydride (0.441 g, 4.36 mmol) andDMAP (0.532 g, eq) followed by TEA (1 ML) were added. The reactionmixture was stirred overnight at room temperature. Its TLC was checkedand the reaction mixture was washed with water and brine. The organiclayer was dried over sodium sulfate and the crude product filteredthrough a small pad of silica gel. The solvent was removed and thismaterial was used for the next reaction. The succinate from the abovereaction was dissolved in anhydrous acetonitrile. HBTU (1.59 g, 4.20mmols) and DIEA (1.10 ml) were added and the mixture was swirled for 5minutes. A polystyrene solid support was added to the reaction mixtureand the mixture was shaken overnight at ambient temperature, the solidsupport was filtered, washed and capped using acetic anhydride/Pymixture. The solid support was again washed with dichloromethane,MeOH/DCM and ether (27.10 g, 55 umol/g).

Example 16: Synthesis of Mono Antennary Alpha Anomer siRNA

Using the conjugate building blocks 104 and 105 described earlier (seescheme 16), RNA is synthesized with the ligands attached to 3′-end ofthe sense strand according to known procedures. This is annealed with anantisense strand. The product is shown below.

Example 17: Synthesis of Trifluoroacetamide Derivative forPost-Synthetic Conjugation

The intermediate compound 8 is prepared as shown in Scheme 17 below.

Synthesis of Compound 2: Z-aminocaproic acid (22.2 g, 82.50 mmols) wasdissolved in DMF (250 mL) and cooled to 0° C. To the solution were addeddiisopropyl ethyl amine (44.4 mL, 275 mmols), HBTU (40.4 g, 106.7mmols), and HOBT (30.0 g, 220 mmols). After stirring under argon for 20minutes at 0° C., 4-hydroxy-1-proline methyl ester hydrochloride (20.0g, 110 mmols) was added and the stirring was continued under argon atroom temperature overnight. The reaction mixture was evaporated todryness. To the residue ethyl acetate (250 mL) was added. The organiclayer was washed with water, saturated sodium bicarbonate, water again,and saturated sodium chloride. The organic layer was dried over sodiumsulfate, filtered and evaporated to dryness. Crude Compound 2 (Rf=0.5 in10% MeOH/DCM, 24.30 g) was obtained. Compound 2 was purified by columnchromatography first by eluting with 2% methanol/dichloromethane toremove impurities followed by 5% methanol/dichloromethane gave 21.36 g(65%). ¹H NMR (400 MHz, DMSO-d₆): Observed rotamers due to amide bond atthe ring. δ 7.35 (m, 5H), 5.15 (d, OH, D₂O exchangeable), 4.99 (s, 2H),4.27 (m, 1H), 3.97 (m, 1H), 3.58 (s, 1H) 3.20-3.47 (m, 5H), 2.94-3.02(m, 2H), 2.10-2.32 (m, 2H), 1.74-2.01 (m, 2H), 1.35-1.4 (m, 4H),1.22-1.28 (m, 4H).

Synthesis of Compound 3: Compound 2 (21.36 g, 54.43 mmols) was dissolvedin THF (200 mL). The reaction mixture was stirred under argon for 20minutes at 0° C. Then lithium borohydride (1.19 g, 54.43 mmols) wasadded to the solution over 20 minutes at 0° C., and the stirring wascontinued under argon at room temperature overnight. The reactionmixture was cooled to 0° C. The excess lithium borohydride was quenchedwhite 5M NaOH (30 mL). After stirring for 30 minutes the reactionmixture was evaporated to dryness. To the residue dichloromethane (200mL) was added. The organic layer was washed with water and saturatedsodium chloride. The organic layer was dried over sodium sulfate,filtered and evaporated to dryness. Crude Compound 3 (Rf=0.4 in 10%MeOH/DCM, 35.88 g) was obtained. Compound 3 was purified by columnchromatography by eluting with 3% methanol/dichloromethane to removeimpurities followed by 5% methanol/dichloromethane to obtain 9.21 g(49%). ¹H NMR (400 MHz, DMSO-d₆): Observed rotamers due to amide bond atthe ring. δ 7.35 (m, 5H), 4.99 (s, H), 4.91 (d, OH, D₂O exchangeable),4.77 (t, OH, D₂O exchangeable), 4.27 (m, 1H), 3.97 (m, 1H), 3.20-3.47(m, 5H), 2.94-3.02 (m, 2H), 2.10-2.32 (m, 2H), 1.74-2.01 (m, 2H),1.35-1.4 (m, 4H), 1.22-1.28 (m, 4H). ¹³C NMR (100 m MHz, DMSO-d₆): δ171.4, 171.1 (minor due to rotamer), 156.1, 137.3, 128.9, 128.3, 128.2,127.7, 125.3, 68.2, 67.4, 65.1, 63.4, 62.0, 57.6, 55.1, 54.9, 53.3,40.1, 39.9, 39.7, 39.5, 39.3, 39.1, 38.9, 37.4, 36.1, 34.2, 32.6, 29.3,26.1, 26.0, 24.6, 24.1, 21.0.

Synthesis of Compound 4: Compound 3 (9.21 g, 25.27 mmols) wasco-evaporated with anhydrous pyridine (80 mL) twice. Then the compoundwas placed under hard vacuum overnight to dry. Compound 3 was taken fromthe hard vacuum and dissolved in anhydrous pyridine (200 mL). To thissolution a catalytic amount of dimethylamino pyridine (0.35 g, 2.53mmols) was added. The reaction mixture stirred under argon for 30minutes at 0° C. Then DMT-Cl (9.0 g, 26.53 mmols) was added to thesolution at 0° C. The mixture stirred under vacuum followed by argon,and stirring was continued under argon at room temperature overnight.The excess DMT-Cl was quenched with the addition of methanol (15 mL).The reaction mixture was evaporated to dryness, and to the residuedichloromethane (200 mL) was added. The organic layer was washed withwater, saturated sodium bicarbonate, water again, and saturated sodiumchloride. The organic layer was dried over sodium sulfate, filtered andevaporated to dryness. Crude Compound 4 (Rf=0.6 in 100% EtOAc, 14.02 g)was obtained. Compound 4 was purified by column chromatography by firsteluting with 50% ethyl acetate (1% TEA) in hexanes to remove impuritiesfollowed by 100% ethyl acetate (1% TEA) to give 12.36 g (73.4%) as awhite foamy solid. ¹H NMR (400 MHz, DMSO-d₆): δ 7.17-7.33 (m, 14H), 4.99(s, 2H), 4.91 (d, OH, D₂O exchangeable), 4.37 (m, 1H), 4.01 (m, 1H),3.72 (s, 6H) 3.56 (m, 1H) 3.29 (m, 1H), 3.14 (m, 1H), 2.93-3.02 (m, 4H),2.18 (m, 2H) 1.74-2.01 (m, 2H), 1.37-1.41 (m, 6H).

Synthesis of Compound 5: Compound 4 (12.36 g, 18.54 mmols) was dissolvedin 10% methanol/ethyl acetate (300 mL) and purged with argon. To thereaction mixture was added 10% palladium by wt. on active carbon wetDegussa type (1.3 g). The flask was re-purged with argon. The flask waspurged with hydrogen twice, then hydrogen was bubbled through thereaction mixture for 10 seconds. The reaction mixture continued to stirunder hydrogen at room temperature overnight. The reaction mixture wasdecanted onto a sintered funnel packed with celite and washed twice withmethanol. The organic layer was evaporated to dryness affording compound5 (Rf=0.05 10% MeOH/DCM, 9.16 g, 93%) as a white solid, which requiredno further purification. ¹H NMR (400 MHz, DMSO-d₆): δ 7.15-7.31 (m, 9H),6.86 (m, 4H) 4.99 (s, 1H), 4.37 (m, 1H), 4.01 (m, 2H), 3.72 (s, 6H) 3.56(m, 1H) 3.29 (m, 1H), 3.14 (m, 1H), 2.93-3.02 (m, 2H), 2.45 (m, 2H),2.18 (m, 2H) 1.74-2.01 (m, 2H), 1.37-1.41 (m, 3H) 1.13-1.38 (m, 4H).

Synthesis of Compound 6: Compound 5 (9.16 g, 17.2 mmols) was dissolvedin dichloromethane (200 mL). The reaction mixture stirred under argonfor 10 minutes at 10° C. To the reaction mixture, triethyl amine (4.80mL, 34.4 mmols) was added dropwise as the mixture continued to stirunder argon for 20 minutes at 10° C. To the reaction mixture ethyltrifluoro acetate (3.05 mL, 25.8 mmols) was added dropwise as themixture continued to stir under argon for 10 minutes at 10° C. Thereaction mixture continued to stir under argon at room temperatureovernight. The reaction mixture was washed with water and saturatedsodium chloride. The organic layer was dried over sodium sulfate,filtered and evaporated to dryness. Crude Compound 6 (Rf=0.6 10%MeOH/DCM, 10.89 g) was obtained. Upon column purification by elutingwith 5% methanol/dichloromethane (1% TEA). Compound 6 (8.76 g, 81%) wasobtained as a yellow foamy solid. ¹H NMR (400 MHz, DMSO-d₆): δ 7.56-7.09(m, 9H), 7.01-6.52 (m, 4H), 5.34-5.04 (m, 1H), 4.99-4.78 (m, 1H),4.48-4.25 (m, 2H), 3.83-3.67 (m, 6H), 3.60-3.50 (m, 1H), 3.49-3.18 (m,2H), 3.16-2.91 (m, 2H), 2.89-2.56 (m, 2H), 2.54-2.32 (m, 2H), 2.32-1.69(m, 3H), 1.59-1.03 (m, 4H). ¹⁹F NMR (400 MHz, DMSO-d₆): −77.14 (s, 3F).

Synthesis of compound 7: Compound 6 (8.76 g, 13.93 mmols), dimethylaminopyridine (5.10 g, 41.79 mmols), and triethyl amine (3.90 mL, 27.86mmols) were dissolved in dichloromethane (300 mL). The reaction mixturewas stirred under argon for 10 minutes. Then succinic anhydride (2.80 g,27.86 mmols) was added and the mixture continued stirring under argon atroom temperature overnight. The reaction mixture was washed with asolution of slightly saturated sodium chloride twice. The organic layerwas dried over sodium sulfate, filtered and evaporated to dryness.Compound 7 (Rf=0.9 10% MeOH/DCM, 10.87 g, 94%) was obtained as a whitesolid, which required no further purification.

Synthesis of Compound 8: Compound 7 (2.00 g, 2.41 mmols) was dissolvedin acetonitrile (100 mL). To the solution, diisopropyl ethyl amine (1.68mL, 9.64 mmols) and HBTU (1.83 g, 4.82 mmols) were added. The reactionmixture was shaken for 10 minutes. CPG (27 g) was added to the flask andthe mixture continued to shake overnight. The CPG compound and reactionmixture were decanted over a sintered funnel. The reaction mixture waswashed with 1% triethyl amine/dichloromethane, followed by two washes of10% methanol/dichloromethane, another wash of 1% triethylamine/dichlormethane, and anhydrous diethyl ether. The CPG compound wassuction dried for 1 hour, then recovered from the funnel and placedunder hard vacuum for 2 hours. CPG compound (5.0 mg) was sent forde-blocking. To the remaining CPG compound was added 25% aceticanhydride/pyridine (100 mL) and the mixture was shaken overnight. TheCPG compound and reaction mixture were placed over a sintered funnel andwashed in the same manner as before. The CPG compound was suction driedfor 1 hour, removed from the funnel and placed under hard vacuum for 2hours. The CPG compound (7.1 mg) was sent for dc-blocking.Spectrophotometer: Before capping 0.9892 Abs (502.0 nm) 65 micromol/g.After capping 1.4403 (502.0 nm) 67 micromol/g.

Synthesis of Compound 9: Compound 6 (8.89 g, 14.14 mmols) anddiisopropyl ethyl amine (4.93 mL, 28.28 mmols) were dissolved inanhydrous dichloromethane (60 mL). The reaction mixture was stirredunder argon for 5 minutes. Then N,N-diisopropylamino cyanoethylphosphoramidic chloride (5.63 mL, 16.26 mmols) was added to the reactionmixture. The reaction mixture continued stirring under argon at roomtemperature for 30 minutes. Completion of the reaction was observed byTLC. The reaction mixture was diluted with dichloromethane (100 mL). Theorganic layer was washed with water, saturated sodium bicarbonate, wateragain, and saturated sodium chloride. The organic layer was dried oversodium sulfate, filtered and evaporated to dryness affording crudeCompound 9 (Rf=0.44 5% MeOH/DCM, 11.02 g). Upon column purification byeluting with 3% methanol/dichloromethane (1% TEA). Compound 9 (6.31 g,54%) was obtained as a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.37(s, 1H), 7.63 (d 1H), 7.42-6.98 (m, 8H), 6.92-6.77 (m, 4H), 4.25-3.90(m, 2H), 3.78-3.64 (m, 7H), 3.48 (d, 3H), 3.29 (d, 1H), 3.23-2.92 (m,4H), 2.86 (d, 1H), 2.73 (t, 1H), 2.58 (t, 1H), 2.53-2.47 (m, 4H),2.33-1.87 (m, 4H), 1.55-0.97 (m, 12H). ³¹P) (400 MHZ, DMSO-d₆): 151.68(d, 1P).

Example 18: Synthesis of Carbamate Linker for Post-Synthetic Conjugation

4-Z-aminobutanol 13: 4-aminobutan-1-ol (26 mL, 280 mmols) andCbz-OSuccinate (104.7 g, 420 mmols) were dissolved in dichloromethane(200 mL). The reaction mixture was stirred under argon for 20 minutes at10° C. Then triethyl amine (78 mL, 560 mmols) was added to the solutionwhile the reaction mixture continued stirring under argon at 10° C. Thereaction mixture continued stirring under argon at room temperatureovernight. The reaction mixture was washed with water, saturated sodiumbicarbonate, water again, and saturated sodium chloride. The organiclayer was dried over sodium sulfate, filtered and evaporated to drynessaffording crude Compound 2 (Rf=0.5 10% MeOH/DCM, 71.97 g) as a whitesolid. This was used in the next step without further purification.

DSC activated 4-Z-aminobutanol 14: Crude Compound 13 (6.0 g) and DSC(10.33 g 40.31 mmols) were dissolved in dichloromethane (100 mL). Thereaction mixture stirred under argon for 30 minutes at 0° C. Triethylamine (7.88 mL, 53.74 mmols) was added dropwise as the mixture stirredunder argon for 5 minutes at 0° C. The reaction mixture continued tostir under argon at room temperature overnight. The reaction mixture wasdiluted in dichloromethane (100 mL). The organic layer was washed withwater, water again, and saturated sodium chloride. The organic layer wasdried over sodium sulfate, filtered and evaporated to dryness affordingcrude Compound 14 (Rf=0.85 10% MeOH/DCM, 11.14 g) as a light brownsolid. This was used in the next step without further purification.

Compound 17: Compound 16 (5.00 g, 7.48 mmols) was dissolved in 10%methanol/ethyl acetate (100 mL) and purged with argon. To the reactionmixture was added 10% palladium by wt. on active carbon wet Degussa type(0.5 g). The flask was re-purged with argon. The flask was purged withhydrogen twice, then hydrogen was bubbled through the reaction mixturefor 10 seconds. The reaction mixture continued to stir under hydrogen atroom temperature overnight. The reaction mixture was decanted onto asintered funnel packed with Celite and washed twice with methanol. Theorganic layer was evaporated to dryness affording Compound 17 (Rf=0.035% MeOH/DCM, 3.4 g, 87%) as a white solid, which required no furtherpurification. ¹H NMR (400 MHz, DMSO-d₆) δ 7.41-7.14 (m, 9H), 6.99-6.72(m, 4H), 4.96 (d, 1H), 4.30 (s, 1H), 4.04-3.58 (m, 6H), 3.46-3.25 (m,1H), 3.21-2.81 (m, 3H), 2.52-2.46 (m, 2H), 1.97 (d, 3H), 1.63-1.42 (m,4H).

Compound 18: Compound 17 (3.2 g, 5.98 mmols) was dissolved indichloromethane (80 mL). The reaction mixture stirred under argon for 10minutes at 10° C. To the reaction mixture, triethyl amine (1.67 mL,11.96 mmols) was added dropwise as the mixture continued to stir underargon for 20 minutes at 10° C. To the reaction mixture, ethyl trifluoroacetate (1.06 mL, 8.97 mmols) was added dropwise as the mixturecontinued to stir under argon for 10 minutes at 10° C. The reactionmixture continued to stir under argon at room temperature overnight. Thereaction mixture was washed with water, saturated sodium bicarbonate,water again, and saturated sodium chloride. The organic layer was driedover sodium sulfate, filtered and evaporated to dryness. Compound 18(Rf=0.45 5% MeOH/DCM, 3.33 g, 88%) was obtained as a yellow foamy solid,which required no further purification. ¹H NMR (400 MHz, DMSO-d₆) δ7.46-7.03 (m, 9H), 6.86 (d, 4H), 4.93 (s, 1H), 4.29 (s, 1H), 4.11-3.63(m, 2H), 3.53-2.79 (m, 5H), 2.53 (d, 2H), 2.30 (d, 2H), 1.99 (d, 3H),1.76 (s, 1H), 1.36 (t, 4H). ¹⁹F NMR (400 MHz, DMSO-d6): −77.11 (s, 3F).

Compound 19: Compound 18 (2.0 g, 3.17 mmols), dimethylamino pyridine(1.16 g, 3.48 mmols), and triethyl amine (0.88 mL, 6.34 mmols) weredissolved in dichloromethane (50 mL). The reaction mixture was stirredunder argon for 10 minutes. Then succinic anhydride (0.63 g, 6.34 mmols)was added and the mixture continued stirring under argon at roomtemperature overnight. The reaction mixture was washed with a solutionof slightly saturated sodium chloride twice. The organic layer was driedover sodium sulfate, filtered and evaporated to dryness. Crude Compound19 (Rf=0.9 10% MeOH/DCM, 3.23 g) was obtained. Upon column purificationby eluting with 5% methanol/dichloromethane (1% TEA). Compound 12 (2.58g, 98%) was obtained as a white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.43(d, 1H), 7.45-7.04 (m, 9H), 7.02-6.71 (m, 4H), 5.51-4.91 (m, 2H),4.16-2.95 (m, 9H), 2.67 (q, 4H), 2.58-2.36 (m, 6H), 2.31-2.02 (m, 2H),1.33 (d, 4H).

Compound 20: Compound 19 (2.03 g, 2.40 mmols) was dissolved inacetonitrile (100 mL). To the solution, diisopropyl ethyl amine (1.67mL, 9.64 mmols) and HBTU (1.82 g, 4.82 mmols) were added. The reactionmixture was shaken for 10 minutes. CPG (27 g) was added to the flask andthe mixture continued to shake overnight. The CPG compound and reactionmixture were decanted over sintered funnel. The reaction mixture waswashed with 1% triethyl amine/dichloromethane, followed by two washes of10% methanol/dichloromethane, another wash of 1% triethylamine/dichlormethane, and anhydrous diethyl ether. The CPG compound wassuction dried for 1 hour, then recovered from the funnel and placedunder hard vacuum for 2 hours. The CPG compound (6.7 mg) was sent forde-blocking. To the remaining CPG compound was added 25% aceticanhydride/pyridine (150 mL) and the mixture was shaken overnight. TheCPG compound and reaction mixture were placed over a sintered funnel andwashed in the same manner as before. The CPG compound was suction driedfor 1 hour, removed from the funnel and placed under hard vacuum for 2hours. The CPG compound (6.9 mg) was sent for de-blocking.Spectrophotometer: Before capping 1.8396 Abs (502.0 nm) 90.3 micromol/g.After capping 1.8798 Abs (502.0 nm) 89.6 micromol/g.

Compound 21: Compound 18 (4.26 g, 6.75 mmols) and diisopropyl ethylamine (2.35 mL, 13.5 mmols) were dissolved in anhydrous dichloromethane(40 mL). The reaction mixture stirred under argon for 5 minutes. ThenN,N-diisopropylamino cyanoethyl phosphoramidic chloride (1.73 mL, 7.76mmols) was added to the reaction mixture. The reaction mixture continuedstirring under argon at room temperature for 30 minutes. Completion ofthe reaction was observed by TLC. The reaction mixture was diluted withdichloromethane (100 mL). The organic layer was washed with water,saturated sodium bicarbonate, water again, and saturated sodiumchloride. The organic layer was dried over sodium sulfate, filtered andevaporated to dryness affording crude Compound 14 (Rf=0.44 5% MeOH/DCM,11.02 g). Upon column purification by eluting with 3%methanol/dichloromethane (1% TEA). Compound 14 (6.31 g, 54%) wasobtained as a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 7.41-6.99 (m,8H), 6.85 (d, 4H), 4.10-3.62 (m, 2H), 3.59-3.24 (m, 7H), 3.13 (d, 3H),2.75-2.68 (m, 1H), 2.59-2.45 (m, 4H), 2.10 (d, 1H), 1.37 (t, 1H),1.25-1.00 (m, 10H).

Example 19: GalNAc Conjugation on Pyrimidines, Purines, and Abasic Sites(Schemes 19-33)

Using palladium coupling chemistry. GalNAc ligands and cationicmolecules can be introduced at the C-5 position of pyrimidinenucleosides with various substituents at the 2¹-position. As shown inschemes below (Scheme 19 Scheme 27), the nucleoside building blocks canbe synthesized accordingly. In Schemes 28 and 29, designed purinenucleoside analogs containing GalNAc ligands are shown. In Scheme 30,GalNAc ligands are introduced at the C-1′ position. Using Clickchemistry, as shown in scheme 31 and 32, GalNAc can be introduced at anabasic site. Scheme 33 shows convertible nucleoside approaches tointroduce GalNAc ligands at the C-2 position on a purine ring.

i. Synthesis of Compound 103 (R═F)

To a solution of 102 (R═F; 11.2 g, 30.1 mmol; Antiviral Chemistry &Chemotherapy. 2010, 21, 15-31) in pyridine (70 mL), was added DMTrCl(11.2 g, 32.1 mmol). The reaction mixture was stirred at roomtemperature for 14 hours and then evaporated. The residue was extractedwith CH₂Cl₂ and saturated NaHCO₃ aq. and dried over anhydrous Na₂SO₄.The crude was purified by silica gel column chromatography (5% MeOH inCH₂Cl₂, R_(f)=0.23) to give compound 103 (17.8 g, 26.4 mmol, 88%). ¹HNMR (DMSO-d₆, 400 MHz): δ 11.81 (s, 1H), 8.07 (s, 1H), 7.46-7.14 (m,10H), 6.88 (dd, J=8.9, 1.8 Hz, 4H), 5.83 (d, J=20.5 Hz, 1H), 5.60 (d,J=7.0 Hz, 1H), 5.16 (dd, J=53.4, 4.8 Hz, 1H), 4.43-4.20 (m, 1H),4.08-3.90 (m, 1H), 3.74 (s, 7H), 3.23 (d, J=2.7 Hz, 2H). ¹³C NMR (100MHz, DMSO-d₆): δ 160.68, 158.09, 158.07, 149.89, 145.13, 144.72, 136.12,135.46, 135.37, 129.71, 127.93, 127.67, 126.70, 123.90, 85.61, 81.30,69.75, 62.47, 55.06, 55.03.

ii. Synthesis of Compound 104 (R═F)

To a solution of compound 103 (R═F; 6.40 g, 9.49 mmol) in CH₃CN (75 mL)were added PdCl₂(PhCN)₂ (73 mg, 0.190 mmol). Et₃N (2.65 mL, 19.0 mmol)and CF₃CH₂OH (6.91 mL, 94.9 mmol). The mixture was stirred at 60° C.under CO gas atmosphere. After evaporation, the residue was extractedwith CH₂Cl₂ and saturated aqueous NaHCO₃. The organic layer was separateand dried over anhydrous Na₂SO₄. The filtrate was concentrated and theresulting crude material was purified by silica gel columnchromatography (0-5% MeOH in CH₂Cl₂) to give 104 (4.30 g, 6.37 mmol,67%, R_(f)=0.32 developed by 5% MeOH in CH₂Cl₂). ¹H NMR (400 MHz,DMSO-d₆): δ 11.78 (s, 1H), 8.52 (s, 1H), 7.41 (d, J=7.4 Hz, 2H),7.34-7.16 (m, 8H), 6.93-6.80 (m, 4H), 5.90 (d, J=20.1 Hz, 1H), 5.63 (d,J=7.1 Hz, 1H), 5.26 (d, J=4.4 Hz, 1H), 5.12 (d, J=4.5 Hz, 1H), 4.50-4.17(m, 3H), 4.07 (dd, J=7.3, 5.0 Hz, 1H), 3.73 (d, J=1.2 Hz, 7H), 3.31-3.17(m, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ 160.70, 158.96, 158.08, 158.06,149.18, 148.89, 144.76, 135.47, 135.33, 129.70, 129.66, 127.80, 127.61,126.66, 124.62, 121.86, 102.57, 94.10, 92.27, 90.68, 90.32, 85.56,81.36, 68.11, 67.95, 62.36, 59.26, 58.91, 54.98. ¹⁹F NMR (376 MHz,DMSO-d₆): δ −74.94, −74.96, −74.99, −201.81, −201.86, −201.87, −201.93,−201.95, −202.01, −202.07. Molecular weight for C₃₃H₃₀F₄N₂NaO₉ (M+Na)⁺Calc. 697.1785. Found 697.2.

iii. Synthesis of Compound 105 (R═F)

Compound 104 (R═F; 4.15 g, 6.15 mmol) was treated with3-(dimethylamino)-1-propylamine (25 mL) at room temperature overnight.The reaction mixture was extracted with CH₂Cl₂ and saturated aqueousNaHCO₃ and the organic layer was dried over anhydrous Na₂SO₄ to givecrude 105. Molecular weight for C₃₆H₄₂FN₄O₈ (M+H)⁺ Calc. 677.2987. Found677.1.

Example 20. GalNAc Conjugation on Ribose Ring (Schemes 34-44)

A synthetic approach to conjugate GalNAc and its derivatives to theribose rings in nucleosides is shown below. Tin-modified nucleosides canbe coupled with alkyl bromides to generate 2′- and 3′-coupled products.Each of the resulting primary amines or activated esters as well asterminal alkenes can be coupled with GalNAc ligands using appropriatereaction conditions. These building blocks are incorporated intooligonucleotides using standard phosphoramidite chemistry. GalNAcligands can also be conjugated with oligonucleotides by post-syntheticapproaches.

2′- and 3′-O-phthalimidohexyl-5-methyluridine (2a, 2b). A solution of2%3%0-dibutylstannylene-5-methyluridine (28 g, 57.24 mmol) obtained asreported (J. Org. Chem., 1974, 24-30), 6-bromohexyl phthalimide (35.5 g,114.48 mmol) and NaI (1.72 g, 11.45 mmol) in DMF (105 mL) was heated at100° C. in a microwave for 3.5 hours. After removing DMF, the residuewas purified by silica gel column chromatography (R_(f)=0.26 in 5% MeOHin CH₂Cl₂) to obtain 2′- and 3′-isomers of theO-phthalmidohexyl-5-methyluridine as an inseparable mixture (10.1 g,20.7 mmol, 36%). MS m/z 488.0 (M+H)⁺, 510.2 (M+Na)⁺, 486.2 (M−H). ¹H NMR(400 MHz, DMSO-d₆): δ 11.29 (s, 1H), 7.88-7.79 (m, 4H), 7.78-7.70 (m,1H), 5.81 (d, J=5.3 Hz, 1H), 5.72 (d, J=5.6 Hz, 1H), 5.25 (d, J=6.2 Hz,1H), 5.12 (t, J=5.0 Hz, 1H), 5.00 (d, J=5.9 Hz, 1H), 4.14 (dd, J=11.3,5.6 Hz, 1H), 4.07 (dd, J=10.1, 5.0 Hz, 1H), 3.89-3.79 (m, 2H), 3.76-3.72(m, 1H), 3.63 (ddd, J=11.1, 8.8, 5.4 Hz, 1H), 3.59-3.47 (m, 4H), 3.41(dt, J=11.6, 6.6 Hz, 1H), 3.28 (s, 1H), 1.75 (d, J=3.7 Hz, 3H),1.64-1.41 (m, 5H), 1.27 (d, J=14.2 Hz, 5H). ¹³C NMR (100 MHz, DMSO-d₆):δ 167.99, 167.97, 163.78, 163.73, 150.79, 150.57, 136.22, 136.07,134.40, 131.60, 131.56, 123.02, 109.38, 109.26, 87.73, 85.89, 85.07,82.71, 80.82, 77.49, 72.43, 69.69, 69.51, 68.38, 60.86, 60.61, 54.92,37.39, 37.35, 29.23, 28.96, 27.93, 27.76, 26.15, 26.07, 25.76, 25.39,25.16, 24.96, 21.27, 12.26.

5′-O-Dimethoxytrityl-2′-O-phthalimidohexyl-5-methyluridine (3a). Themixture of 2a and 2b (10.11 g, 20.73 mmol) was co-evaporated withpyridine (50 mL) then dissolved in pyridine (80 mL) and cooled to 0° C.in an ice bath. To this mixture, dimethoxytrityl chloride (7.73 g, 22.80mmol) was added and reaction stirred at 0° C. for 2 hours then at roomtemperature for 1 hour. An additional 0.4 eq of dimethoxytrityl chloridewas added and reaction stirred overnight. Reaction quenched with MeOH (5mL) then evaporated in vacuo. The reaction mixture was diluted with DCMand washed with brine twice. The organic layer was dried with Na₂SO₄ andevaporated in vacuo, then purified via silica gel column chromatographyto yield 4.48 g of 3a (27.4%, 5.67 mmol; Rf=0.35 in 60% EtOAc inhexane). MS m/z 812.3 (M+Na)⁺, 788.3 (M−H). ¹H NMR (400 MHz, DMSO-d₆): δ11.35 (s, 1H), 7.87-7.77 (m, 4H), 7.48 (d, J=0.8 Hz, 1H), 7.38 (d, J=7.4Hz, 2H), 7.30 (t, J=7.6 Hz, 2H), 7.23 (dd, J=12.1, 8.1 Hz, 5H), 6.89 (d,J=8.0 Hz, 4H), 5.11 (d, J=6.3 Hz, 1H), 4.18 (dd, J=11.1, 5.4 Hz, 1H),3.99 (ddd, J=12.0, 11.2, 5.9 Hz, 3H), 3.72 (s, 6H), 3.62-3.46 (m, 4H),3.21 (ddd, J=12.9, 10.7, 3.3 Hz, 2H), 1.98 (d, J=1.8 Hz, 1H), 1.60-1.44(m, 5H), 1.38 (s, 3H), 1.35-1.19 (m, 5H), 1.16 (t, J=7.1 Hz, 1H), 0.87(dd, J=10.1, 4.7 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆): δ 170.33, 167.92,163.61, 158.19, 158.16, 150.41, 144.67, 135.45, 135.32, 135.11, 134.33,131.58, 129.74, 127.93, 127.65, 126.84, 122.97, 113.27, 109.60, 86.46,85.91, 83.09, 80.53, 69.63, 68.76, 63.48, 63.19, 59.75, 55.06, 37.31,30.16, 28.89, 27.92, 26.07, 24.95, 20.76, 20.72, 18.60, 14.08, 13.55,11.66.

5′-O-Dimethoxytrityl-3′-O-phthalimidohexyl-5-methyluridine (3b). The3′-isomer (3b) was isolated by column chromatography (3.36 g, 20.5%,4.26 mmol; R_(f)=0.18 in 60% EtOAc in hexane). MS m/z 812.0 (M+Na)⁺,788.3 (M−H). ¹H NMR (400 MHz, DMSO-d₆): δ 11.34 (s, 1H), 7.88-7.76 (m,4H), 7.50 (s, 1H), 7.36 (d, J=7.5 Hz, 2H), 7.33-7.15 (m, 7H), 6.87 (d,J=7.8 Hz, 4H), 5.75-5.69 (m, 1H), 5.37 (d, J=6.0 Hz, 1H), 4.27 (dd,J=10.5, 5.1 Hz, 1H), 4.05-3.94 (m, 2H), 3.91 (t, J=5.2 Hz, 1H), 3.71 (s,6H), 3.56 (ddd, J=22.7, 11.8, 6.7 Hz, 3H), 3.21 (ddd, J=24.1, 10.8, 3.3Hz, 2H), 1.98 (t, J=4.5 Hz, 1H), 1.60-1.37 (m, 7H), 1.34-1.13 (m, 5H),0.94-0.83 (m, 1H), ¹³C NMR (100 MHz, DMSO-d₆): δ 170.41, 167.93, 163.69,158.17, 158.16, 150.57, 144.63, 135.73, 135.30, 135.17, 134.35, 131.58,129.71, 127.91, 127.63, 126.82, 122.97, 113.24, 109.37, 88.69, 85.91,80.60, 77.27, 72.18, 69.67, 63.49, 62.95, 59.76, 55.03, 54.90, 37.33,30.17, 29.09, 27.88, 26.08, 25.08, 20.76, 20.72, 18.87, 18.61, 14.09,13.55, 11.74.

5′-O-Dimethoxytrityl-2′-O-aminohexyl-5-methyluridine (4a). 3a (15.0 g,18.99 mmol) was dissolved in 190 mL MeOH. Hydrazine (3.04 g, 94.52 mmol)was added to heterogeneous mixture and heated to reflux (66° C.) for 3.5hours. The mixture was cooled to room temperature and evaporated invacuo to yield a white powder. The product was dissolved in DCM andwashed with ammonium hydroxide and saturated NaCl solution twice. TheDCM layer was dried with MgSO₄ and evaporated in vacuo to yield 11.8 g.The crude material was used for next step. (R_(f)=0.02 in 5% MeOH inDCM). MS m/z 660.2 (M+H)⁺, 682.1 (M+Na)⁺, 658.1 (M−H)⁻. ¹H NMR (400 MHz,DMSO-d₆): δ 7.48 (s, 1H), 7.38 (d, J=7.5 Hz, 2H), 7.30 (t, J=7.5 Hz,2H), 7.24 (d, J=8.9 Hz, 5H), 6.89 (d, J=8.4 Hz, 4H), 5.84 (d, J=5.0 Hz,1H), 5.74 (s, 1H), 4.19 (t, J=5.0 Hz, 1H), 3.97 (t, J=4.9 Hz, 2H), 3.72(s, 6H), 3.63-3.45 (m, 3H), 3.27-3.15 (m, 3H), 1.48 (d, J=6.4 Hz, 2H),1.38 (s, 3H), 1.34-1.18 (m, 6H).

5′-O-Dimethoxytrityl-2′-O-aminohexyl-C5-GalNAc(O-Bz)-5-methyluridine(5a). Crude 4a (6.00 g) was dissolved in DCM (250 mL) and triethylamine(3.8 mL, 27.30 mmol) and stirred for 10 minutes. GalNAc-C5-NHS ester(7.31 g, 10.0 mmol) was added and the reaction mixture was stirred for 2hours. The reaction mixture was diluted with DCM and washed with brinethen the organic layer was dried with Na₂SO₄ and evaporated in vacuo.The crude was purified via column chromatography (R_(f)=0.36 in 5% MeOHin DCM) to yield 5a (9.56 g, 7.49 mmol, 83%). MS m/z 1298.3 (M+Na)⁺. ¹HNMR (400 MHz, DMSO-d₆): δ 11.36 (s, 1H), 7.99-7.86 (m, 5H), 7.73-7.52(m, 8H), 7.48 (t, J=7.7 Hz, 3H), 7.41-7.34 (m, 4H), 7.30 (t, J=7.6 Hz,2H), 7.23 (dd, J=11.1, 8.1 Hz, 4H), 6.88 (d, J=8.1 Hz, 4H), 5.83 (d,J=4.8 Hz, 1H), 5.74 (d, J=3.9 Hz, 1H), 5.35 (dd, J=11.1, 3.3 Hz, 1H),5.11 (d, J=6.3 Hz, 1H), 4.72 (d, J=8.5 Hz, 1H), 4.50-4.39 (m, 2H),4.38-4.14 (m, 4H), 3.95 (t, J=4.8 Hz, 2H), 3.82-3.68 (m, 7H), 3.63-3.43(m, 4H), 3.28-3.15 (m, 3H), 2.98 (dd, J=12.9, 6.5 Hz, 2H), 2.03 (s, 2H),1.69 (s, 3H), 1.49 (s, 6H), 1.40-1.15 (m, 9H), ¹³C NMR (100 MHz,DMSO-d₆): δ 171.73, 169.40, 165.20, 165.16, 164.86, 163.62, 158.18,158.15, 150.41, 144.64, 135.31, 135.10, 133.50, 129.73, 129.16, 129.00,128.70, 128.59, 127.91, 127.64, 126.82, 113.26, 109.59, 100.89, 85.91,83.09, 80.60, 71.86, 69.97, 69.74, 68.77, 67.92, 63.19, 62.03, 55.04,49.74, 35.03, 29.17, 29.04, 28.59, 26.25, 25.12, 22.69, 21.85, 11.66.

5′-O-Dimethoxytrityl-2′-O-aminohexyl-C5-GalNAc(O-Bz)-3′-O-succinate-5-methyluridine(6a). 5a (2.00 g, 1.57 mmol) was dissolved in DCM (50 mL). DMAP (574 mg,4.70 mmol) and succinic anhydride (313 mg, 3.14 mmol) were added andreaction mixture stirred for 17 hours at room temperature. Product waspurified via silica gel column chromatography (ϕ=4.2 cm×15 cm) treatedwith 2% TEA in DCM. Product was eluted with 0-5% MeOH and 2-5% TEA inDCM and co-evaporated with acetonitrile in vacuo to yield 2.11 g (91%,1.43 mmol) of 6a as a TEA salt (R_(f)=0.41 in 5% MeOH/5% TEA in DCM). MSm/z 1397.4 (M+Na)⁺, 1373.4 (M−H)⁻. ¹H NMR (400 MHz, DMSO-d₆): δ 11.44(s, 1H), 8.11 (d, J=9.0 Hz, 1H), 7.90 (dd, J=10.2, 4.1 Hz, 4H), 7.75 (s,1H), 7.72-7.52 (m, 7H), 7.51-7.44 (m, 3H), 7.34 (ddd, J=24.0, 13.7, 7.9Hz, 6H), 7.22 (d, J=8.7 Hz, 5H), 6.89 (d, J=7.9 Hz, 4H), 5.83 (d, J=6.1Hz, 1H), 5.73 (d, J=3.4 Hz, 1H), 5.36 (dd, J=11.1, 3.3 Hz, 1H),5.26-5.22 (m, 1H), 4.75 (d, J=8.5 Hz, 1H), 4.47-4.40 (m, 2H), 4.37-4.22(m, 3H), 4.12 (d, J=3.5 Hz, 1H), 3.83-3.69 (m, 6H), 3.53-3.19 (m, 15H),2.97 (d, J=7.8 Hz, 2H), 2.04 (s, 2H), 1.68 (s, 2H), 1.56-1.11 (m, 15H).¹³C NMR (100 MHz, DMSO-d₆): δ 173.40, 171.76, 171.63, 169.39, 165.20,165.15, 164.86, 163.50, 158.22, 150.50, 144.48, 135.12, 134.95, 133.48,129.70, 129.16, 129.03, 128.70, 128.58, 127.95, 127.61, 113.30, 110.16,100.91, 86.13, 69.97, 67.91, 62.04, 55.04, 49.73, 45.48, 40.12, 39.92,39.71, 39.50, 39.29, 39.08, 38.87, 38.33, 34.98, 29.13, 28.93, 26.19,25.08, 22.68, 21.82, 11.69, 10.48.

5%0-Dimethoxytrityl-2′-O-aminohexyl-C5-GalNAc(O-Bz)-3′-O-CPG-5-methyluridine(7a). To a solution of 6a (2.01 g, 1.36 mmol) in acetonitrile (100 mL)was added HBTU (1.03 g, 2.72 mmol), DIEA (528 mg, 4.08 mmol) and CPG(16.0 g, 130 μmol/g, 540 Å) and the mixture was shaken for 24 hours. CPGwas filtered out and washed with DCM, 20% MeOH in DCM, then ether. ThenCPG was treated with acetic anhydride (25 mL) in pyridine (75 mL) andTEA (1 mL) for 1 hour. CPG was filtered out and washed with the samesolvents above. Loading was measured twice with a spectrophotometer andaverage loading calculated (73.1 μmol/g).

5′-O-Dimethoxytrityl-2′-O-aminohexyl-C5-GalNAc(O-Bz)-3′-O—(N,N-diisopropyl)-β-cyanoethylphosphoramidite-5-methyluridine(8a). 5a (2.90 g, 2.27 mmol) was co-evaporated with anhydrousacetonitrile twice then put under a strict argon atmosphere. 5a wasdissolved in anhydrous DCM (35 mL) and cooled to 0° C.2-cyanoethyl-N,N,N′N′-tetraisopropylphosphordiamidite (1.37 g, 4.55mmol) was added to stilling mixture followed by DCI (268 mg, 2.27 mmol).Mixture stirred at 0° C. for 20 minutes then room temperature for 17hours. Reaction mixture was diluted with DCM, then washed with brine,and dried with Na₂SO₄ to give a pale yellow foam. The crude was purifiedvia silica gel column chromatography (ϕ=4.2 cm×19 cm; R_(f)=0.39 inEtOAc) to yield 3.20 g of 8a (95%, 2.17 mmol). MS m/z 1498.3 (M+Na)⁺. ¹HNMR (400 MHz, DMSO-d₆): δ 11.39 (s, 1H), 8.06-7.84 (m, 5H), 7.78-7.16(m, 21H), 6.93-6.82 (m, 4H), 5.82 (d, J=4.3 Hz, 1H), 5.74 (s, 1H), 5.35(dd, J=11.1, 3.1 Hz, 1H), 4.73 (d, J=8.5 Hz, 1H), 4.50-4.20 (m, 5H),4.10 (dd, J=12.8, 7.7 Hz, 2H), 3.74 (t, J=11.5 Hz, 7H), 3.66-3.44 (m,6H), 3.29-3.18 (m, 2H), 2.98 (d, J=5.3 Hz, 2H), 2.76 (t, J=5.8 Hz, 1H),2.57 (dd, J=10.0, 5.3 Hz, 1H), 2.49 (d, J=1.5 Hz, 5H), 2.04 (s, 2H),1.70 (s, 3H), 1.59-1.03 (m, 29H), 0.99-0.81 (m, 4H). ¹³C NMR (125 MHz,DMSO-d₆) δ 171.60, 169.26, 165.07, 165.03, 164.73, 163.48, 158.10,158.07, 150.24, 150.22, 144.39, 144.34, 135.32, 135.08, 134.99, 134.97,134.87, 134.80, 133.64, 133.37, 133.34, 129.65, 129.07, 129.03, 128.90,128.88, 128.87, 128.83, 128.56, 128.45, 127.75, 127.56, 127.50, 126.74,118.72, 118.57, 113.08, 109.63, 109.54, 100.76, 87.02, 86.62, 85.92,85.90, 82.13, 79.89, 71.72, 69.84, 68.61, 67.78, 62.57, 62.23, 61.89,58.43, 57.81, 57.65, 54.91, 49.60, 46.05, 45.53, 42.42, 34.88, 29.02,28.97, 28.45, 26.17, 25.04, 24.19, 24.13, 24.03, 22.55, 21.71, 11.50.³¹P NMR (162 MHz, DMSO-d₆) δ 154.01, 153.65.

5%0-Dimethoxytrityl-3′-O-aminohexyl-5-methyluridine (4b). 3b (3.64 g,4.61 mmol) was dissolved in MeOH. Hydrazine (738 mg, 23.04 mmol) wasadded and reaction mixture refluxed for 5.5 hours. The same procedureswere followed as described for 4a to yield 2.93 g of crude 4b,(R_(f)=0.02 in 5% MeOH in DCM). MS m/z 660.2 (M+H)⁺, 682.1 (M+Na)⁺,658.1 (M−H)⁻. ¹H NMR (400 MHz, DMSO-d₆) δ 7.50 (s, 1H), 7.43-7.16 (m,9H), 6.88 (d, J=8.7 Hz, 4H), 5.73 (d, J=4.6 Hz, 1H), 4.29 (t, J=4.8 Hz,1H), 4.02-3.89 (m, 3H), 3.46-3.15 (m, 5H), 2.24-2.16 (m, 1H), 2.05 (s,1H), 1.58-1.10 (m, 13H). ¹³C NMR (100 MHz, DMSO-d₆): δ 163.73, 158.04,158.02, 150.58, 144.49, 135.54, 135.18, 135.06, 129.56, 127.78, 127.50,126.69, 113.11, 109.23, 88.49, 85.78, 80.47, 77.20, 72.02, 69.62, 62.84,54.91, 41.44, 33.12, 29.17, 26.24, 26.11, 25.31, 11.62.

5′-O-Dimethoxytrityl-3′-O-aminohexyl-C5-GalNAc(O-Bz)-5-methyluridine(5b). To a solution of 4b (2.85 g, 4.32 mmol) in DCM (45 mL) and TEA(1.8 mL) was added GalNAc-NHS ester (3.47 g, 4.75 mmol). Reactionstirred for 2 hours before additional 0.2 eq of GalNAc-NHS ester wasadded then stirred an additional 1 hour. The same procedures werefollowed as described for 5a to yield 3.62 g of Sb (2.84 mina 66%).(R_(f)=0.24 in 5% MeOH in DCM). MS m/z 1297.4 (M+Na)⁺. ¹H NMR (400 MHz,DMSO-d₆): δ 11.35 (s, 1H), 8.02-7.87 (m, 5H), 7.74-7.44 (m, 11H),7.41-7.34 (m, 4H), 7.33-7.19 (m, 7H), 6.88 (d, J=8.8 Hz, 4H), 5.74 (dd,J=5.9, 4.1 Hz, 2H), 5.41-5.33 (m, 2H), 4.73 (d, J=8.5 Hz, 1H), 4.44 (t,J=7.9 Hz, 2H), 4.31 (ddd, J=16.9, 12.6, 7.9 Hz, 3H), 4.03-3.95 (m, 1H),3.91 (t, J=5.1 Hz, 1H), 3.83-3.75 (m, 1H), 3.72 (s, 6H), 3.63-3.45 (m,2H), 3.22 (dd, J=7.8, 2.9 Hz, 2H), 2.99 (d, J=6.2 Hz, 2H), 2.87 (s, 1H),2.72 (s, 1H), 2.04 (t, J=6.4 Hz, 2H), 1.69 (s, 3H), 1.55-1.14 (m, 14H).¹³C NMR (125 MHz, DMSO-d₆): δ 171.74, 169.40, 165.20, 165.16, 164.86,163.65, 162.27, 158.16, 150.57, 144.58, 135.70, 135.32, 135.17, 133.74,133.45, 129.68, 129.15, 129.01, 128.94, 128.67, 128.57, 127.89, 127.63,126.81, 113.23, 109.37, 100.89, 88.58, 85.91, 80.64, 77.36, 72.17,71.84, 69.98, 69.73, 68.74, 67.92, 62.98, 62.03, 55.02, 54.85, 49.75,40.33, 40.05, 39.77, 39.49, 39.21, 38.94, 38.66, 38.34, 35.74, 35.02,30.74, 29.23, 29.12, 28.58, 26.26, 25.21, 22.66, 21.84, 11.68.

5′-O-Dimethoxytrityl-3′-O-aminohexyl-C5-GalNAc(O-Bz)-2′-O-succinate-5-methyluridine(6b). To a solution of 5b (1.10 g, 0.86 mmol) in DCM (20 mL) was addedDMAP (315 mg, 2.58 mmol) and succinic anhydride (172 mg, 1.73 mmol).Reaction mixture was stirred for 23 hours then purified in similarmanner to 6a and co-evaporated with acetonitrile in vacuo to yield 1.03g of 6b (81%, 0.70 mmol). (R_(f)=0.18 in 5% MeOH in DCM). MS m/z 1397.4(M+Na)⁺, 1373.4 (M−H)⁻. ¹H NMR (400 MHz, DMSO-d₆): δ 8.09 (d, J=9.3 Hz,1H), 7.91 (t, J=6.9 Hz, 4H), 7.76 (t, J=5.4 Hz, 1H), 7.69 (dd, J=11.3,7.5 Hz, 3H), 7.65-7.51 (m, 5H), 7.47 (t, J=7.7 Hz, 2H), 7.41-7.33 (m,4H), 7.32-7.16 (m, 7H), 6.87 (d, J=8.7 Hz, 4H), 5.85 (d, J=3.9 Hz, 1H),5.74 (d, J=3.2 Hz, 1H), 5.48-5.41 (m, 1H), 5.36 (dd, J=11.1, 3.2 Hz,1H), 5.29 (s, 1H), 4.75 (d, J=8.5 Hz, 1H), 4.49-4.39 (m, 2H), 4.38-4.19(m, 4H), 3.28 (ddd, J=37.9, 12.7, 5.4 Hz, 11H), 3.08-2.90 (m, 3H), 2.69(q, J=7.2 Hz, 5H), 2.60-2.52 (m, 2H), 2.05 (d, J=3.5 Hz, 2H), 1.76 (s,1H), 1.69 (s, 3H), 1.56-0.94 (m, 28H). ¹³C NMR (100 MHz, DMSO-d₆): δ173.63, 173.44, 172.79, 171.92, 171.52, 169.54, 165.29, 165.24, 164.94,163.78, 158.23, 150.33, 144.57, 136.18, 135.27, 135.18, 133.84, 133.58,129.77, 129.24, 129.05, 128.76, 128.65, 127.96, 127.70, 126.90, 113.29,109.73, 100.98, 85.97, 80.75, 73.31, 71.96, 70.48, 62.40, 55.10, 52.06,51.36, 45.45, 35.04, 33.35, 29.20, 29.09, 28.95, 28.77, 22.70, 21.89,11.81, 10.05, 7.26, 7.17.

5′-O-Dimethoxytrityl-3′-O-aminohexyl-C5-GalNAc(O-Bz)-2′-O-CPG-5-methyluridine(7b). To a solution of 6b (970 mg, 0.66 mmol) in acetonitrile (50 mL)was added HBTU (497 mg, 1.31 mmol). DIEA (339 mg, 1.97 mmol) and CPG(8.20 g, 130 μmol/g, 540 Å) and the mixture was shaken for 21 hours. CPGwas filtered, washed, capped and measured in the same manner as 7a toyield CPG 7b with an average loading of 56.1 μmol/g.

5′-O-Dimethoxytrityl-3′-O-aminohexyl-C5-GalNAc(O-Bz)-2′-O-(cyanoethylN,N-diisopropyl)-phosphoramidite-5-methyluridine (8b). 5b (1.98 g, 1.55mmol) was treated in the same manner described for 8a and silica gelcolumn chromatography gave 1.89 g (83%, 1.28 mmol) of 8b. (R_(f)=0.43 in100% EtOAc). MS m/z 1497.4 (M+Na)⁺, 1474.3 (M−H)⁻. ¹H NMR (400 MHz,DMSO-d₆): δ 11.38 (s, 1H), 8.00-7.87 (m, 5H), 7.75-7.18 (m, 22H), 6.88(dd, J=8.9, 2.7 Hz, 4H), 5.88 (dd, J=9.2, 5.1 Hz, 1H), 5.75 (d, J=3.2Hz, 1H), 5.36 (dd, J=11.1, 3.2 Hz, 1H), 4.73 (d, J=8.5 Hz, 1H),4.61-4.50 (m, 1H), 4.49-4.40 (m, 2H), 4.38-4.23 (m, 2H), 4.01 (ddd,J=19.3, 11.6, 4.8 Hz, 3H), 3.85-3.47 (m, 14H), 2.98 (s, 2H), 2.71 (ddd,J=11.6, 8.9, 3.9 Hz, 2H), 2.10-1.95 (m, 4H), 1.69 (s, 3H), 1.57-0.99 (m,31H), 0.94-0.83 (m, 2H). ¹³C NMR (125 MHz, DMSO-d₆): δ 171.72, 169.37,165.18, 165.14, 164.85, 163.58, 163.50, 158.18, 158.16, 150.48, 150.43,144.52, 135.15, 134.98, 133.74, 133.48, 133.44, 129.66, 129.14, 129.01,128.99, 128.98, 128.93, 128.67, 128.56, 127.91, 127.88, 127.57, 126.84,118.81, 118.71, 113.25, 113.21, 109.72, 109.68, 100.86, 86.17, 86.02,71.82, 69.95, 69.86, 68.72, 67.88, 63.45, 62.00, 55.00, 49.72, 39.80,39.63, 39.46, 39.30, 39.13, 38.96, 38.33, 35.00, 30.12, 30.03, 29.26,29.14, 29.11, 28.56, 26.29, 25.27, 25.25, 24.28, 24.22, 24.08, 24.02,22.65, 21.84, 20.67, 19.80, 19.75, 19.67, 19.62, 18.56, 14.04, 13.50,11.68, 11.55. ³¹P NMR (162 MHz, DMSO-d₆) δ 155.08, 154.60.

Example 21. Synthesis of S- and C-Linked GalNAc Derivatives and BuildingBlocks (Schemes 45-51)

Syntheses of Compounds (Schemes 45-51)

Compound 1 (15.6 g, 40.1 mmol) was treated with TMSOTf (7.98 mL, 44.1mmol) in DCE to give compound 2. Molecular weight for C₁₄H₂₀NO₈ (M+H)⁺Calc. 330.12. Found 330.0.

Synthesis of compound 3: Compound 2 (1.65 g, 5 mmol) and tert-butyl5-mercaptopentanoate (1.0 g, 5.25 mmol) in DCE were treated with TMSOTf(0.181 mL, 1.0 mmol) overnight. Aqueous work-up and silica gel columnpurification gave compound 3 (380 mg, 0.731 mmol, 15%). Molecular weightfor C₂₃H₃₇NNaO₁₀S (M+H)⁺ Calc 542.20. Found 542.1.

Synthesis of compound 7: To a solution of compound 3 (380 mg, 0.731mmol) in CH₂Cl₂ (4 mL) was added TFA (1 mL) at 0° C. The mixture wasstirred at 0° C. for 2 h then at room temperature for 3 h. The solventwas evaporated and the residue was co-evaporated with toluene to givecrude compound 7. This material was used for next step withoutpurification. Molecular weight for C₁₉H₃₀NO₁₀S (M+H)⁺ Calc 464.1590.Found 464.1.

Synthesis of compound 8: Compound 7 from the previous step (˜0.731 mmol)was treated with N-hydroxysuccinimide (168 mg, 1.46 mmol) in thepresence of EDCI (280 mg, 1.46 mmol) and DMA (0.764 mL, 4.38 mmol) inCH₂Cl₂ (5 mL) for 14 h. Aqueous work-up then column chromatography gavecompound 8 (284 mg, 0.507 mmol, 69% over 2 steps). Molecular weight forC₂₃H₃₃N₂O₁₂S (M+H)⁺ Calc. 561.1754. Found 561.1.

S-alkylation of compound 6 with alkyne bromide gives compound 9. In asimilar way, compound 14 is prepared from compound 11 (J. Org. Chem.,2002, 67, 2995-2999). The acid 12 is prepared according to the reportedprocedure.

Synthesis of compound 13: Compound 12 (2.40 g, 5.18 mmol) was treatedwith N-hydroxysuccinimide (716 mg, 6.22 mmol) in the presence of EDCI(1.19 g, 6.22 mmol) and DIEA (2.70 mL, 15.5 mmol) in CH₂Cl₂ (30 mL) for14 h. Aqueous work-up then column chromatography gave compound 13 (1.83g, 3.26 mmol, 63%). Molecular weight for C₂₃H₃₃N₂O₁₂S (M+H)⁺ Calc.561.1754. Found 561.2.

Compound 16 was prepared using reported procedures (3. Org. Chem., 2006,71, 3619-3622; Carbohydrate Research, 1998, 309, 319-330).

Synthesis of compound 22: Compound 16 (1.94 g, 5.22 mmol), benzyl4-pentenoate (2.99 g, 15.7 mmol) and Grubbs Catalyst, 2nd Generation(433 mg, 0.522 mmol) in CH₂Cl₂ (20 mL) were heated at 40° C. for 40 h.The solvent was removed and the residue was purified by silica gelcolumn chromatography to give compound 22 (1.87 g, 3.50 mmol, 67%).Molecular weight for C₂₇H₃₆NO₁₀ (M+H)⁺ Calc. 534.2339. Found 534.2.

Synthesis of compound 23: To a solution of compound 22 (1.85 g, 3.47mmol) in EtOAc (30 mL) was added palladium on carbon (Aldrich:330108-50G, 10 wt. %, Degussa type E101 NE/W: 185 mg). The reactionmixture was stirred under H₂ atmosphere for 14 h. After filtrationthrough Celite, the filtrate was removed in vacuo. The residue waspurified by silica gel column chromatography to give compound 23 (903mg, 2.03 mmol, 59%). Molecular weight for C₂₀H₃₂NO₁₀ (M+H)⁺ Calc.446.2026. Found 446.1.

Synthesis of compound 24: Compound 23 (326 mg, 0.732 mmol) was treatedwith N-hydroxysuccinimide (127 mg, 1.10 mmol) in the presence of EDCI(211 mg, 1.10 mmol) and DIEA (0.383 mL, 2.20 mmol) in CH₂Cl₂ (5 mL) for14 h. Aqueous work-up then column chromatography gave compound 24 (300mg, 0.553 mmol, 76%). Molecular weight for C₂₄H₃₅N₂O₁₂ (M+H)⁺ Calc.543.2190. Found 543.2.

Oxidative cleavage of 16 gives aldehyde 25. It is reduced to an alcoholand subjected to O-alkylation with a benzyl-protected triflate followedby deprotection and esterification to give 28. Reductive amination of 25gives acid 29 and is followed by esterification to give 30.

Compound 35 can be synthesized in a similar way reported in theliterature (J. Org. Chem., 1996, 61, 6442-6445). As described above forScheme 48, compounds 38, 42, and 44 are prepared.

Activated esters 8, 13, 24, 38, 28, 42, 30, 44 are coupled withtriamine- (45) or monoamine- (48) containing hydroxyprolinol to give 46and 49, respectively. These compounds are converted to theircorresponding phosphoramidites or loaded onto solid supports.

DMTr-protected di-azide 53 is coupled with alkyne-containing GalNAcderivatives using Click chemistry to give compound 54. It is convertedto its corresponding phosphoramidite or loaded onto solid support toyield 55.

Example 22. Synthesis of C2-derivatized Galactosamine Analogs for ASGPRBinding (Schemes 52-53)

C2-derivatized galactosamine analogs may be prepared as shown in Schemes52 and 53 below.

Compound 58 is prepared in a similar way to reported procedures (see,e.g., WO96/39411). O-alkylation followed by selective cleavage of acetylgroups gives compound 60 and 63. The NHS esters 61 and 64 are preparedby a standard esterification. Acetyl groups of 65 are removedselectively and the resulting hydroxyl groups are protected by benzylgroups to give 66. Oxidative cleavage of terminal alkene gives 67.Esterification followed by hydrogenation gives 61/64.

Trifluoromethyl acetamide- (TFA-) protected galactosamine (GalN-TFA) NHSesters are coupled with amine-containing oligonucleotides (69/71) togenerate Gal-TFA containing oligonucleotides (70/72) in a post-syntheticapproach.

Example 23. Synthesis of ASGPR Ligand Mimics Containing PseudouridineScaffold (Schemes 55-56)

Psuedouridine ligands may be prepared as shown in Schemes 55 and 56.

Compound 452: To a solution of pseudouridine 451 (20 g, 81.9 mmol) in 1Mtriethylammoniumbicarbonate buffer (pH 8.5, 780 mL) and EtOH (940 mL),methyl acrylate (235 mL, 2.61 mol) was added dropwise. The reactionmixture was stirred for 16 hours. After removal of the solvent, thecrude material was purified by silica gel column chromatography (10%MeOH in CH₂Cl₂, R_(f)=0.23) to give compound 452 (26.6 g, 80.5 mmol,98%). ¹H NMR (MeOH-d₄, 400 MHz): δ 7.77 (d, J=0.8 Hz, 1H), 4.58 (d,J=4.8 Hz, 1H), 4.15 (t, J=5.2 Hz, 1H), 4.05 (t, J 5.0 Hz, 1H), 3.98-4.02(m, 2H), 3.91-3.94 (m, 1H), 3.80 (dd, J=12.0 Hz, 3.3 Hz, 1H), 3.67 (s,3H), 3.66 (dd, J=12.0 Hz, 3.3 Hz, 1H), 2.73-2.77 (m, 2H). ¹³C NMR(CDCl₃, 100 MHz): δ 173.1, 165.4, 152.5, 145.8, 112.9, 85.6, 81.5, 75.6,72.6, 63.3, 52.5, 46.2, 33.7. MW for C₁₃H₁₉N₂O₈ (M+H)⁺ Calc. 330.11.Found 331.0.

Compound 453: To a solution of compound 452 (11.67 g, 35.3 mmol) in DMF(65 mL), di-tert-butylsilyl bis(trifluoromethanesulfonate) (15.46 mL,414 mmol) was added dropwise under stirring at 0° C. The reactionmixture was kept stirring at 0° C. for 30 min and treated with imidazole(12.0 g, 176.5 mmol). The mixture was stirred at 0° C. for 10 min andthen at room temperature for 30 min. TBDMSCl (7.98 g, 53.0 mmol) wasadded and the reaction mixture was heated at 75° C. for 6 hours. Thereaction mixture was extracted with Et₂O and saturated NaHCO₃ aq., driedover anhydrous Na₂SO₄, and concentrated. The residue was purified bysilica gel column chromatography (hexane:EtOAc=1:1, R_(f)=0.50) to givecompound 453 (15.0 g, 25.6 mmol, 73%). ¹H NMR (DMSO-d₆, 400 MHz): δ11.39 (s, 1H), 7.54 (s, 1H), 4.55 (s, 1H), 4.34-4.38 (m, 1H), 4.18 (d,J=4.4 Hz, 1H), 3.86-4.00 (m, 5H), 3.58 (s, 3H), 2.67 (t, J=6.6 Hz, 2H),1.02 (s, 9H), 0.99 (s, 9H), 0.89 (s, 9H), 0.13 (s, 3H), 0.087 (s, 3H).MW for C₂₇H₄₉N₂O₈Si₂ (M+H)⁺ Calc. 585.30. Found 585.2.

Compound 454: Compound 453 (1.24 g, 2.12 mmol) was treated withethylenediamine (10 mL) at room temperature for 2 hours. Ethylenediaminewas removed by evaporation and the residue was dried in vacuo. The crudewas extracted with CH₂Cl₂ and saturated NaHCO₃ aq., dried over anhydrousNa₂SO₄, and concentrated to give 454 as a white solid (1.16 g, 1.89mmol, 89%). ¹H NMR (MeOD-d₄, 400 MHz): δ 7.49 (s, 1H), 4.63 (s, 1H),4.39-4.41 (m, 1H), 4.29 (d. J=3.6 Hz, 1H), 4.00-4.04 (m, 5H), 3.18-3.26(m, 2H), 2.69 (t, J=6.2 Hz, 2H), 2.56-2.61 (m, 2H), 1.07 (s, 9H), 1.04(s, 9H), 0.94 (s, 9H), 0.17 (s, 3H), 0.13 (s, 3H). MW for C₂₈H₅₃N₄O₇Si₂(M+H)⁺ Calc. 61135. Found 613.2.

Compound 455: To a solution of GalNAc acid (930 mg, 2.08 mmol) in DMF(10 mL), HBTU (789 mg, 2.08 mmol) and iPr₂NEt (1.65 mL, 9.45 mmol) wereadded. After 10 min, compound 454 in DMF (15 mL) was added to thesolution and stirred overnight. The reaction mixture was extracted withEt₂O and saturated NaHCO₃ aq. and dried over anhydrous Na₂SO₄. Afterevaporation, the crude was purified by silica gel column chromatography(10% MeOH in CH₂Cl₂, R_(f)=0.43) to give compound 455 (1.83 g, 1.76mmol, 93%). ¹H NMR (DMSO-d₆, 400 MHz): δ 11.36 (s, 1H), 7.98 (s, 1H),7.82 (d, J=9.2 Hz, 1H) 7.77, (s, 1H), 7.51 (s, 1H), 5.21 (d, J=3.6 Hz,1H), 4.96 (dd, J=11.4 Hz, 3.4 Hz, 1H), 4.53 (s, 1H), 4.48 (d, J=8.4 Hz,1H), 4.33-4.36 (m, 1H), 4.18 (d, J=4.4 Hz, 1H), 3.85-4.02 (m, 9H),3.67-3.73 (m, 1H), 3.37-3.43 (m, 1H), 3.04 (s, 4H), 2.39-2.44 (m, 2H),2.10 (s, 3H), 2.02-2.05 (m, 2H), 1.99 (s, 3H), 1.89 (s, 3H), 1.77 (s,3H), 1.46-1.49 (m, 4H), 1.01 (s, 9H), 0.99 (s, 9H), 0.89 (s, 9H), 0.12(s, 3H), 0.080 (s, 3H). ¹³C NMR (DMSO-d₆, 100 MHz): δ 172.0, 169.8,169.7, 169.5, 169.2, 162.3, 150.3, 143.4, 110.6, 100.8, 83.4, 76.2,74.7, 73.1, 70.3, 69.7, 68.5, 67.5, 66.6, 61.3, 54.9, 54.8, 49.3, 44.6,38.3, 38.0, 34.9, 33.9, 28.5, 27.3, 26.7, 25.7, 25.6, 22.6, 22.0, 21.6,20.4, 20.3, 19.8, 17.8, −4.5, −5.1. MW for C₄₇H₇₉N₅NaO₁₇Si₂ (M+Na)⁺Calc. 1064.49. Found 1064.2.

Compound 456: Hydrogen fluoride-pyridine (˜70% HF, 0.165 mL, 6.34 mmol)was diluted in pyridine (2 mL) under cooling. The resulting solution wasadded to a solution of compound 455 in CH₂Cl₂ at 0° C., and the mixturewas stirred at 0° C. for 2 hours. The reaction solution was diluted inCH₂C₁₁, washed with saturated NaHCO₃ aq., and dried over anhydrousNa₂SO₄. After evaporation, the crude was dried in memo to give a whitefoam. To a solution of this material in pyridine (15 mL). DMTrCl (596mg, 1.76 mmol) was added. The reaction mixture was stirred at roomtemperature for 4 hours and then evaporated. The residue was extractedwith CH₂Cl₂ and saturated NaHCO₃ aq, and dried over anhydrous Na₂SO₄.The crude was purified by silica gel column chromatography (10% MeOH inCH₂Cl₂, R_(f)=0.57) to give compound 456 (1.65 g, 1.37 mmol, 78%). ¹HNMR (DMSO-d₆, 400 MHz): δ 11.33 (s, 1H), 7.92 (s, 1H), 7.81 (d, J=9.6Hz, 1H), 7.75, (s, 1H), 7.42-7.44 (m, 3H), 7.19-7.32 (m, 7H), 6.87-6.90(m, 4H), 5.21 (d, J=3.2 Hz, 1H), 4.96 (dd, J=11.4 Hz, 3.4 Hz, 1H), 4.63(d, J=6.4 Hz, 1H), 4.53 (d, J=2.4 Hz, 1H), 4.48 (d, J=8.4 Hz, 1H),4.02-4.07 (m, 4H), 3.81-3.91 (m, 3H), 3.73 (s, 6H), 3.68-3.70 (m, 2H),3.53-3.63 (m, 1H), 3.23-3.40 (m, 2H), 3.02-3.14 (m, 5H), 2.32-2.35 (m,2H), 2.10 (s, 3H), 2.00-2.04 (m, 2H), 1.99 (s, 3H), 1.89 (s, 3H), 1.76(s, 3H), 1.44-1.47 (m, 4H), 0.87 (s, 9H), 0.064 (s, 3H), 0.041 (s, 3H).MW for C₆₀H₈₁N₅NaO₁₉Si (M+Na)⁺ Calc. 1226.52. Found 1226.4.

Compound 457: To a solution of compound 456 (1.86 g, 1.54 mmol) inCH₂Cl₂ (20 mL), 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite(1.47 mL, 4.63 mmol) and 4,5-dicyanoimidazole (182 mg, 1.54 mmol) wereadded at 0° C. The reaction mixture was stirred at room temperature for20 hours under argon atmosphere. The reaction mixture was diluted withCH₂Cl₂ (300 mL) and washed with saturated NaHCO₃ (100 mL). The organiclayer was separate and dried over anhydrous Na₂SO₄. The filtrate wasconcentrated and the resulting crude material was purified by silica gelcolumn chromatography (EtOAc then 0-3% MeOH in CH₂Cl₂) to give 457 (1.80g, 1.28 mmol, 83%, R_(f)=0.43 developed by 10% MeOH in CH₂Cl₂). ¹H NMR(400 MHz, DMSO-d₆): δ 11.34 (s, 0.5H), 11.33 (s, 0.5H), 7.91 (s, 1H),7.81 (d, J=9.2, 1H), 7.75 (s, 1H), 7.56 (s, 0.5H), 7.52 (s, 0.5H), 7.43(t, J=8.2, 2H), 7.19-7.32 (m, 7H), 6.85-6.90 (m, 4H), 5.21 (s, 0.5H),5.21 (s, 0.5H), 4.96 (dd, J=11.2, 3.4, 1H), 4.47-4.51 (m, 2H), 4.36-4.41(m, 1H), 4.02-4.07 (m, 5H), 3.83-3.90 (m, 1H), 3.73 (s, 3H), 3.72 (s,3H), 3.69-3.71 (m, 3H), 3.31-3.60 (m, xx H), 3.04-3.26 (m, 6H),2.69-2.73 (m, 1H), 2.35 (t, J=6.3, 2H), 2.10 (s, 3H), 2.03 (m, 2H), 1.99(s, 3H), 1.89 (s, 3H), 1.77 (s, 3H), 1.45-1.48 (m, 4H), 0.91-1.08 (m,12H), 0.85 (s, 9H), 0.063 (s, 1.5H), 0.046 (s, 1.5H), 0.035 (3H). ³¹PNMR (DMSO-d₆, 162 MHz) δ 147.92, 147.70. MW for C₆₉H₉₈N₇NaO₂₀PSi (M+Na)⁺Calc. 1426.63. Found 1426.5.

Compound 458: To a solution of 452 (21.5 g, 65.1 mmol) in pyridine (400mL), DMAP (1.59 g, 13.0 mmol) and DMTrCl (22.1 g, 65.1 mmol) were added.The reaction mixture was stirred at room temperature for 6 hours andthen evaporated. The residue was extracted with EtOAc and saturatedNaHCO₃ aq., dried over anhydrous Na₂SO₄, and purified by silica gelcolumn chromatography (5% MeOH in CH₂Cl₂, R_(f)=0.30) to give 458 (36.2g, 57.2 mmol, 88%). ¹H NMR (DMSO-d₆, 400 MHz): δ 11.37 (s, 1H), 7.48 (s,1H), 7.36 (d, J=8.0 Hz, 2H), 7.27-7.32 (m, 6H), 7.20-7.23 (m, 1H),6.87-6.90 (m, 4H), 5.06 (d, J=4.8 Hz, 1H), 4.80 (d, J=6.4 Hz, 1H), 4.54(d, J=2.8 Hz, 1H), 3.84-3.93 (m, 1H), 3.73 (s, 6H), 3.56-3.69 (m, 2H),3.53 (s, 3H), 3.15-3.17 (m, 2H), 2.58 (t, J=6.6 Hz, 2H). ¹³C NMR(MeOH-d₄, 100 MHz): δ 172.7, 165.5, 160.2, 152.7, 146.4, 144.5, 137.4,137.3, 131.5, 131.4, 129.6, 128.9, 128.0, 114.2, 114.0, 87.5, 83.0,81.1, 76.2, 72.4, 64.7, 55.8, 52.4, 46.2, 33.5. MW for C₃₄H₃₆N₂NaO₁₀(M+Na)⁺ Calc. 655.23. Found 655.2.

Compound 459: Compound 458 (13.9 g, 22.0 mmol) was treated withethylenediamine (75 mL) at room temperature for 18 hours.Ethylenediamine was removed by evaporation and co-evaporated withtoluene. The residue was extracted with CH₂Cl₂/MeOH (180 mL/20 mL) andH₂O (50 mL) and the organic layer was dried over anhydrous Na₂SO₄, andthen concentrated. The crude was crystallized with hexane and CH₂Cl₂ togive 459 as a pale yellow solid (11.7 g, 17.7 mmol, 80%). ¹H NMR(MeOD-d₄, 400 MHz): δ 7.57 (s, 1H), 7.21-7.48 (m, 9H), 6.86-6.88 (m,4H), 4.71 (d, J=3.2 Hz, 1H), 4.02-4.17 (m, 3H), 3.79-3.82 (m, 1H), 3.78(s, 6H), 3.31-3.36 (m, 3H), 3.15 (t, J=6.2 Hz, 2H), 2.63 (I, J=6.0 Hz,2H), 2.41 (t, J=6.2 Hz, 2H). MW for C₃₅H₄₀N₄NaO₉ (M+H)⁺ Calc. 683.27.Found 683.2.

Compound 460: To a solution of GalNAc acid (5.60 g, 12.5 mmol) in DMF(50 mL). HBTU (4.70 g, 12.4 mmol) and iPr₂NEt (10.3 mL, 59.3 mmol) wereadded. After 10 min, compound 459 in DMF (50 mL) was added to thesolution and stirred overnight. The reaction mixture was extracted withEtOAc and H₂O and dried over anhydrous Na₂SO₄. After evaporation, thecrude was purified by silica gel column chromatography (10% MeOH inCH₂Cl₂, R_(f)=0.50) to give compound 460 (6.85 g, 6.28 mmol, 59%). ¹HNMR (DMSO-d₆, 400 MHz): δ 11.33 (s, 1H), 7.93 (s, 1H), 7.81 (d, J=9.2Hz, 1H), 7.75. (s, 1H), 7.41-7.44 (m, 3H), 7.27-7.31 (m, 6H), 7.18-7.22(m, 1H), 6.87-6.89 (m, 4H), 5.21 (d, J=3.2 Hz, 1H), 5.03 (d, =4.8 Hz,1H), 4.96 (dd, J=11.2 Hz, 3.6 Hz, 1H), 4.78 (d, J=6.4 Hz, 1H), 4.51 (d,J=2.8 Hz, 1H), 4.48 (d, J=8.4 Hz, 1H), 4.02 (m, 3H), 3.82-3.92 (m, 4H),3.73 (s, 6H), 3.54-3.70 (m, 3H), 3.36-3.42 (m, 1H), 3.02-3.21 (m, 6H),2.35 (t, J=6.6 Hz, 2H), 2.09 (s, 3H), 2.02 (1, J=7.0 Hz, 2H), 1.99 (s,3H), 1.88 (s, 3H), 1.76 (s, 3H), 1.43-1.49 (m, 4H). ¹¹C NMR (DMSO-d₆,100 MHz): δ 172.0, 169.9, 169.8, 169.5, 169.4, 169.2, 162.6, 157.9,150.3, 144.9, 143.2, 135.7, 135.6, 129.7, 127.7, 126.5, 113.0, 111.3,100.9, 85.2, 80.7, 79.8, 73.5, 70.8, 70.4, 69.7, 68.5, 66.6, 64.1, 61.3,54.9, 54.8, 49.3, 48.5, 44.8, 38.3, 38.1, 34.9, 33.9, 28.5, 22.7, 21.6,20.4, 20.3. MW for C₅₄H₆₇N₅NaO₁₉ (M+Na)⁺ Calc. 1112.43. Found 1112.2.

Compound 461: To a solution of compound 460 (1.55 g, 1.42 mmol) inpyridine (10 mL), TBDMSCl (214 mg, 1.42 mmol) and imidazole (290 mg,4.26 mmol) were added. The reaction mixture was stirred overnight. Afterevaporation, the residue was extracted with CH₂Cl₂ and saturated NaHCO₃aq, and dried over anhydrous Na₂SO₄. The crude material was purified bysilica gel column chromatography (5% MeOH in CH₂Cl₂, R_(f)=0.15) to givecompound 461 (550 mg, 0.457 mmol, 32%) and its 2′-O-TBDMS isomer 456(390 mg, 0.324 mmol, 23%). ¹H NMR (DMSO-d₆, 400 MHz): δ 11.32 (s, 1H),7.94 (s, 1H), 7.82 (d, J=9.2 Hz, 1H), 7.75, (s, 1H), 7.54 (s, 1H),7.40-7.41 (m, 2H), 7.21-7.32 (m, 7H), 6.87-6.89 (m, 4H), 5.21 (d, J=3.2Hz, 1H), 4.96 (dd, J=11.2 Hz, 3.6 Hz, 1H), 4.73 (d, J=4.8 Hz, 1H),4.47-4.49 (m, 2H), 3.95-4.02 (m, 5H), 3.83-3.88 (m, 2H), 3.72 (s, 6H),3.68-3.71 (m, 3H), 3.38-3.41 (m, 1H), 3.03-3.19 (m, 6H), 2.39 (t, J=6.6Hz, 2H), 2.10 (s, 3H), 2.02 (t, J=7.0 Hz, 2H), 1.99 (s, 3H), 1.89 (s,3H), 1.77 (s, 3H), 1.45-1.50 (m, 4H), 0.74 (s, 9H), −0.034, (s, 3H),−0.11 (s, 3H). MW for C₆₀H₈₁N₅NaO₁₉Si (M+Na)⁺ Calc. 1226.52. Found1227.4.

Compound 462: To a solution of compound 461 (2.28 g, 1.89 mmol) inCH₂Cl₂ (60 mL), DMAP (693 mg, 5.67 mmol) and succinic anhydride (378 mg,3.78 mmol) were added. The reaction mixture was stirred overnight atroom temperature. Silica gel column chromatography (10% MeOH/10% Et₃N inCH₂Cl₂, R_(f)=0.44) of the crude mixture without aqueous work-up gavethe compound 462 as the corresponding triethylammonium salt (2.50 g,1.78 mmol, 94%). ¹H NMR (DMSO-d₆, 400 MHz): δ 8.42 (s, 1H), 8.18 (s,1H), 8.05 (d, J=9.2 Hz, 1H), 7.71 (s, 1H), 7.48-7.50 (m, 2H), 7.29-7.40(m, 7H), 6.95-6.97 (m, 4H), 5.28-5.30 (m, 2H), 5.07 (dd, J=11.2 Hz, 3.6Hz, 1H), 4.70 (d, J=4.0 Hz, 1H), 4.60 (d, J=8.4 Hz, 1H), 4.37 (t, J=5.8Hz, 1H), 4.09-4.13 (m, 3H), 3.91-3.97 (m, 2H), 3.81 (s, 6H), 3.78-3.85(m, 3H), 3.42-3.49 (m, 2H), 3.27-3.30 (m, 1H), 3.10-3.16 (m, 5H),2.43-2.53 (m, 5H), 2.18 (s, 3H), 2.12 (1, J=7.2 Hz, 2H), 2.07 (s, 3H),1.97 (s, 3H), 1.85 (s, 3H), 1.52-1.57 (m, 4H), 0.79 (s, 9H), 0.00 (s,3H), −0.075 (s, 3H). ¹³C NMR (DMSO-d₆, 100 MHz): δ 173.8, 172.2, 172.1,171.5, 169.9, 169.6, 169.5, 169.3, 162.5, 158.1, 150.4, 144.7, 144.6,135.5, 135.4, 129.7, 127.7, 126.6, 113.1, 109.5, 100.9, 85.6, 81.6,77.5, 74.2, 71.0, 70.5, 69.8, 68.5, 66.7, 63.6, 61.4, 52.0, 49.3, 38.4,38.2, 34.9, 34.0, 30.0, 29.5, 28.5, 25.8, 25.5, 25.4, 22.7, 21.6, 21.4,20.5, 20.4, 17.5, 14.7, 7.1, −5.1, −5.4. MW for C₆₄H₈₄N₅O₂₂Si (M−H)⁻Calc. 1302.54. Found 1302.4.

Compound 463: To a solution of compound 462 (98 mg, 0.07 mmol) in DMF(10 mL), HBTU (30 mg, 0.077 mmol), iPr₂NEt (0.061 mL, 0.35 mmol), andaminomethyl polystyrene support (ARTVISION, considered as 70 μmol/g,1.10 g, 0.077 mmol) were successively added. The mixture was shaken for24 hours, then filtered, washed with CH₂Cl₂, and dried in vacuo. Theresidual amino groups were capped by shaking for 1 hour with pyridine(15 mL), acetic anhydride (5 mL), and triethylamine (1 mL). Afterfiltering, washing with CH₂Cl₂ (1.00 mL), then 50% MeOH/CH₂Cl₂ (100 mL),and drying in vacuo gave compound 463 (1.12 g), Loading: 47 μmol/g.

Example 24. Synthesis of ASGPR Ligand Mimics Containing N-GlycosidicLinkage (Schemes 57-63)

ASGPR ligands containing N-glycosidic linkages can be prepared as shownin Schemes 57-63.

Example 25. ASGPR Ligand Mimics (Schemes 64-73)

The ASGPR ligands below can be prepared as shown in Schemes 64-73.

Example 26. Amino linkers for conjugation of ligand to oligonucleotides(Schemes 74-76)

Amino linkers for conjugating ligands to oligonucleotides can beprepared by Schemes 74-76 below.

Example 27. ASGPR Ligand Mimics—Carbohydrate Scaffold

The ASGPR ligand below can be prepared as shown in Scheme 77.

Example 28. Conjugation of GalNAc Ligand to C2 of Purine Base

GalNAc ligands can be prepared to the C2 position of a purine base asshown in Scheme 78 below.

Q^(A) and Q^(B) are any of the ASGPR ligands described herein.

Example 29 siRNA—Ligand Conjugates

RNA Synthesis and Duplex Annealing

1. Oligonucleotide Synthesis

All oligonucleotides were synthesized on an AKTAoligopilot synthesizeror an ABI 394 synthsizer. Commercially available controlled pore glasssolid support (dT-CPG, 500{acute over (Å)}. Prime Synthesis) and RNAphosphoramidites with standard protecting groups. 5′-O-dimethoxytritylN6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite(Pierce Nucleic Acids Technologies) were used for the oligonucleotidesynthesis unless otherwise specified. The 2′-F phosphoramidites,5′-O-dimethoxytrityl-N4-acetyl-2′-fluoro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite and5′-O-dimethoxytrityl-T-fluoro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditewere purchased from (Promega). All phosphoramidites were used at aconcentration of 0.2M in acetonitrile (CH₃CN) except for guanosine whichwas used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recyclingtime of 16 minutes was used. The activator was 5-ethyl thiotetrazole(0.75M, American International Chemicals), for the PO-oxidationIodine/Water/Pyridine was used and the PS-oxidation PADS (2%) in2,6-lutidine/ACN (1:1 v/v) was used.

Ligand conjugated strands were synthesized using a solid supportcontaining the corresponding ligand. For example, the introduction of acarbohydrate moiety/ligand (for e.g., GalNAc) at the 3′-end of asequence was achieved by starting the synthesis with the correspondingcarbohydrate solid support. Similarly a cholesterol moiety at the 3′-endwas introduced by starting the synthesis on the cholesterol support. Ingeneral, the ligand moiety was tethered to brans-4-hydroxyprolinol via atether of choice as described in the previous examples to obtain ahydroxyprolinol-ligand moiety. The hydroxyprolinol-ligand moiety wasthen coupled to a solid support via a succinate linker or was convertedto phosphoramidite via standard phosphitylation conditions to obtain thedesired carbohydrate conjugate building blocks. Fluorophore labeledsiRNAs were synthesized from the corresponding phosphoramidite or solidsupport, purchased from Biosearch Technologies. The oleyl lithocholic(GalNAc)₃ polymer support made in house at a loading of 38.6 μmot/gram.The Mannose (Man)₃ polymer support was also made in house at a loadingof 42.0 μmol/gram.

Conjugation of the ligand of choice at the desired position, for exampleat the 5′-end of the sequence, was achieved by coupling of thecorresponding phosphoramidite to the growing chain under standardphosphoramidite coupling conditions unless otherwise specified. Anextended 15 minute coupling of 0.1M solution of phosphoramidite inanhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activatorto a solid bound oligonucleotide. Oxidation of the internucleotidephosphite to the phosphate was carried out using standard iodine-wateras reported in (1) Beaucage, S. L. (2008) Solid-phase synthesis of siRNAoligonucleotides. Curr. Opin. Drug Discov. Devel., 11, 203-216; (2)Mueller. S., Wolf. J, and Ivanov. S. A. (2004) Current Strategies forthe Synthesis of RNA. Curr. Org. Synth., 1, 293-307 and (3) Xia, J.,Noronha, A., Toudjarska. I., Li. F., Akine, A., Braich. R.,Frank-Kamenetsky. M., Rajeev, K. G., Egli. M, and Manoharan. M. (2006)Gene Silencing Activity of siRNAs with a Ribo-difluorotoluyl Nucleotide.ACS Chem. Biol., 1, 176-183 or by treatment with ten-butylhydroperoxide/acetonitrile/water (10:87:3) with a 10 minute oxidationwait time conjugated oligonucleotide. Phosphorothioate was introduced bythe oxidation of phosphite to phosphorothioate by using a sulfurtransfer reagent such as DDTT (purchased from AM Chemicals). PADS and orBeaucage reagent. The cholesterol phosphoramidite was synthesized inhouse, and used at a concentration of 0.1 M in dichloromethane. Couplingtime for the cholesterol phosphoramidite was 16 minutes.

2. Deprotection—I (Nucleobase Deprotection)

After completion of synthesis, the support was transferred to a 100 mlglass bottle (VWR). The oligonucleotide was cleaved from the supportwith simultaneous protection of base and phosphate groups with 80 mL ofa mixture of ethanolic ammonia [ammonia: ethanol (3:1)] for 6.5 h at 55°C. The bottle was cooled briefly on ice and then the ethanolic ammoniamixture was filtered into a new 250 ml bottle. The CPG was washed with2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixturewas then reduced to ˜30 ml by roto-vap. The mixture was then frozen ondry ice and dried under vacuum on a speed vac.

3. Deprotection-II (Removal of 2′ TBDMS Group)

The dried residue was resuspended in 26 ml of triethylamine,triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6)and heated at 60° C. for 90 minutes to remove theCert-butyldimethylsilyl (TBDMS) groups at the T position. The reactionwas then quenched with 50 ml of 20 mM sodium acetate and pH adjusted to6.5, and stored in freezer until purification.

4. Analysis

The oligonucleotides were analyzed by high-performance liquidchromatography (HPLC) prior to purification and selection of buffer andcolumn depends on nature of the sequence and or conjugated ligand.

5. HPLC Purification

The ligand conjugated oligonucleotides were purified by reverse phasepreparative HPLC. The unconjugated oligonucleotides were purified byanion-exchange HPLC on a TSK gel column packed in house. The bufferswere 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mMsodium phosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractionscontaining full-length oligonucleotides were pooled, desalted, andlyophilized. Approximately 0.15 OD of desalted oligonucleotides werediluted in water to 150 ml and then pipetted in special vials for CGEand LC/MS analysis. Compounds were finally analyzed by LC-ESMS and CGE.

6. RNAi Agent Preparation

For the preparation of RNAi agent, equimolar amounts of sense andantisense strand were heated in 1×PBS at 95° C. for 5 minutes and slowlycooled to room temperature. The integrity of the duplex was confirmed byHPLC analysis. Table 1 below reflects the synthesized RNAi agents.

Example 30: Synthesis of siRNA-Ligand Conjugates Using Post-SyntheticMethods

The single stranded oligonucleotides containing desired amino linkerswere synthesized using the corresponding amino linker monomerscompatible with solid phase oligonucleotide synthesis and deprotectionconditions as described in Example 30 (Schemes 79). After deprotectionthe amino linked oligonucleotides were reacted with NHS esters of theligand shown in the Table below the Scheme 79 followed by treatment withammonia and HPLC purification. Each purified ligand-conjugated singlestranded oligonucleotide was annealied with equimolar mixture ofcomplementary strand yielded the siRNAs shown in Table 5. The (1+1+1)design shown in Scheme 79 and in Table 5 was obtained by coupling ofamino linker phosphoramidite to the amino linker solid supportsuccessively (two synthesis cycle) followed by successive coupling ofnucleoside phosphoramidite monomers as described in Example 29.

Post Synthetic Conjugation of Ligand to Oligonucleotides

The TTR siRNA in each conjugate was the same. Similarly, the AT3 siRNAin each conjugate was the same. The following ligands were attached tothe 3′ end of the sense strand of each siRNA. The left side of theligand description indicates the site of attachment to the 3′ terminusof the sense strand.

TABLE 1 siRNA-Ligand conjugates Conjugate Target Sequence 5′-3′ 43527TTR L96 60148 TTR L193L193L193 60146 TTR s(T3gs)(T3gs)(T3g) 60142 TTRs(Tgs)(Tgs)(Tg) 60133 TTR L199L199L199 60139 TTR L204L204L204 60134 TTRL200L200L200 60132 TTR L198L198L198 60125 TTR L207L207L207 60124 TTRL203L203L203 60122 TTR L203 60123 TTR L206 60135 TTR L207 60136 TTR L20860129 TTR L198 60126 TTR L197 60131 TTR L200 60127 TTR L202 60130 TTRL199 60128 TTR L201 60137 TTR L204 60138 TTR L205 60140 TTR (Tg)(Tg)(Tg)60144 TTR (T3g)(T3g)(T3g) 60141 TTR (Tg) 60143 TTR s(Tg) 60145 TTR (T3g)60147 TTR s(T3g) 58036 TTR L194L194L194 58037 TTR L195L195L195 58038 TTRL194 is on the 3^(th), 4^(th), and 5^(th) nucleotide from the 3′ end.58039 TTR L195 is on the 3^(th), 4^(th), and 5^(th) nucleotide from the3′ end. 58138 TTR L193L193L193 (but different modified antisense strand)55727 TTR L96 on different siRNA AfaCfaGfuGfuUfCfUfuGfcUfcUfaUfaAfL9670001 AT3 L96 70002 AT3 L193L193L193

Example 31 In Vitro Screening of RNAi Agents

Cell Culture and Transfections

Human Hep3B cells or rat H.II.4.E cells (ATCC. Manassas, Va.) were grownto near confluence at 37° C. in an atmosphere of 5% CO2 in RPMI (ATCC)supplemented with 10% FBS, streptomycin, and glutamine (ATCC) beforebeing released from the plate by trypsinization. Transfection wascarried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of LipofectamineRNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl ofsiRNA duplexes per well into a 96-well plate and incubated at roomtemperature for 15 minutes. 80 μl of complete growth media withoutantibiotic containing ˜2×104 Hep3B cells were then added to the siRNAmixture. Cells were incubated for either 24 or 120 hours prior to RNApurification. Single dose experiments were performed at 10 nM and 0.1 nMfinal duplex concentration and dose response experiments were done using8, 4 fold serial dilutions with a maximum dose of 10 nM final duplexconcentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12)

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer thenmixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (themixing speed was the same throughout the process). Ten microliters ofmagnetic heads and 80 μl lysis/Binding Buffer mixture were added to around bottom plate and mixed for 1 minute. Magnetic beads were capturedusing magnetic stand and the supernatant was removed without disturbingthe beads. After removing the supernatant, the lysed cells were added tothe remaining beads and mixed for 5 minutes. After removing thesupernatant, magnetic beads were washed 2 times with 150 μl Wash BufferA and mixed for 1 minute. Beads were capture again and supernatantremoved. Beads were then washed with 150 μl Wash Buffer B, captured andsupernatant was removed. Beads were next washed with 150 μl ElutionBuffer, captured and supernatant removed. Beads were allowed to dry for2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for5 minutes at 70° C., Beads were captured on magnet for 5 minutes. 40 μlof supernatant was removed and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif. Cat #4368813)

A master mix of 1 μl 10× Buffer, 0.4 μl 25× dNTPs, 1 μl Random primers,0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 1.6 μl of H₂Oper reaction were added into 5 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C., 10 min, 37° C. 120 min, 85° C. 5 sec. 4° C.hold.

Real Time PCR

2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqManProbe (Applied Biosystems Cat #4326317E (human) Cat #4308313 (rodent)).0.5 μl TTR TaqMan probe (Applied Biosystems cat #HS00174914_m1 (human)cat #Rn00562124_m1 (rat)) and 5 μl Lightcycler 480 probe master mix(Roche Cat #04887301001) per well in a 384 well plate (Roche cat#04887301001). Real time PCR was done in a Roche LC 480 Real Time PCRmachine (Roche). Each duplex was tested in at least two independenttransfections and each transfection was assayed in duplicate, unlessotherwise noted.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with 10 nM AD-1955, or mock transfected cells. IC₅₀s werecalculated using a 4 parameter fit model using XLFit and normalized tocells transfected with non-target specific control or nave cells overthe same dose range, or to its own lowest dose. IC₅₀s were calculatedfor each individual transfection as well as in combination, where asingle IC₅₀ was fit to the data from both transfections.

Example 32: In Vitro Silencing Activity of Chemically Modified RNAiAgents that Target TTR

The following experiments demonstrated the beneficial effects ofchemical modifications, including the introduction of triplet repeatmotifs, together with a GalNAc₃ ligand, on the silencing activity ofRNAi agents that target TTR.

Protocol for Assessment of IC₅₀ in Hep3B Cells

The IC₅₀ for each modified siRNA was determined in Hep3B cells bystandard reverse transfection using Lipofectamine RNAiMAX. In brief,reverse transfection was carried out by adding 5 μL of Opti-MEM to 5 μLof siRNA duplex per well into a 96-well plate along with 10 μL ofOpti-MEM plus 0.5 μL of Lipofectamine RNAiMax per well (Invitrogen,Carlsbad Calif. cat #13778-150) and incubating at room temperature for15-20 minutes. Following incubation, 100 μL of complete growth mediawithout antibiotic containing 12,000-15,000 Hcp3B cells was then addedto each well. Cells were incubated for 24 hours at 37° C. in anatmosphere of 5% CO₂ prior to lysis and analysis of TTR and GAPDH mRNAby bDNA (Quantigene). Seven different siRNA concentrations ranging from10 nM to 0.6 pM were assessed for IC₅₀ determination and TTR/GAPDH forsiRNA transfected cells was normalized to cells transfected with 10 nMLuc siRNA.

Protocol for Assessment of Free-Uptake IC₅₀

Free uptake silencing in primary cynomolgus or mouse hepatocytes wasassessed following incubation with TTR siRNA for 4 hours or 24 hours.Silencing was measured at 24 hours from the initial exposure.

Example 33: TTR mRNA Silencing and TTR Protein Suppression in Mice

To assess the efficacy of the RNAi agents, these agents wereadministered to mice. The RNAi agents or PBS control were administeredto mice in a single subcutaneous dose of 5 mg/kg or 1 mg/kg. Afterapproximately 48 hours, mice were anesthetized with 200 μl of ketamine,and then exsanguinated by severing the right caudal artery. Whole bloodwas isolated and plasma was isolated and stored at −80° C., untilassaying. Liver tissue was collected, flash-frozen and stored at −80°C., until processing.

Efficacy of treatment was evaluated by (i) measurement of TTR mRNA inliver at 48 and 144 hours post-dose, and (ii) measurement of TTR proteinin plasma at prebleed and at 48/144 hours post-dose. TTR liver mRNAlevels were assayed utilizing the Branched DNA assays-QuantiGene 2.0(Panomics cat #: QS0011). Briefly, mouse liver samples were ground andtissue lysates were prepared. Liver lysis mixture (a mixture of 1 volumeof lysis mixture, 2 volume of nuclease-free water and 10 ul ofProteinase-K/ml for a final concentration of 20 mg/ml) was incubated at65° C. for 35 minutes. 20 μl of Working Probe Set (TTR probe for genetarget and GAPDH for endogenous control) and 80 ul of tissue-lysate werethen added into the Capture Plate. Capture Plates were incubated at 55°C.±1° C. (aprx. 16-20 hrs). The next day, the Capture Plates were washed3 times with 1× Wash Buffer (nuclease-free water. Buffer Component 1 andWash Buffer Component 2), then dried by centrifuging for 1 minute at 240g, 100 μl of pre-Amplifier Working Reagent was added into the CapturePlate, which was sealed with aluminum foil and incubated for 1 hour at55° C.±1° C. Following 1 hour incubation, the wash step was repeated,then 100 μl of Amplifier Working Reagent was added. After 1 hour, thewash and dry steps were repeated, and 100 μl of Label Probe was added.Capture plates were incubated 50° C.±1° C. for 1 hour. The plate wasthen washed with 1× Wash Buffer, dried and 10(411 Substrate was addedinto the Capture Plate. Capture Plates were read using the SpectraMaxLuminometer following a 5 to 15 minute incubation, bDNA data wereanalyzed by subtracting the average background from each triplicatesample, averaging the resultant triplicate GAPDH (control probe) and TTR(experimental probe) values, and then computing the ratio; (experimentalprobe-background)/(control probe-background).

Plasma TTR levels were assayed utilizing the commercially available kitaccording to manufacturer's guidelines. Briefly, mouse plasma wasdiluted 1:10,000 in 1× mix diluents and added to pre-coated plates alongwith kit standards, and incubated for 2 hours at room temperaturefollowed by 5× washes with kit wash buffer. Fifty microliters ofbiotinylated prealbumin antibody was added to each well and incubatedfor 1 hr at room temperature, followed by 5× washes with wash buffer.Fifty microliters of streptavidin-peroxidase conjugate was added to eachwell and plates were incubated for 30 minutes at room temperaturefollowed by washing as previously described. The reaction was developedby the addition of 501.11/well of chromogen substrate and incubation for10 minutes at room temperature with stopping of reaction by the additionof 50 μl/well of stop solution. Absorbance at 450 nm was read on aVersamax microplate reader (Molecular Devices, Sunnyvale, Calif.) anddata were analyzed utilizing the Softmax 4.6 software package (MolecularDevices).

The results of representative siRNA-conjugate efficacy is shown inFIG. 1. Most of anomeric linkage modified ligands showed similar TTRprotein suppression at 5 mg/kg dose.

Example 34: AT3 mRNA Silencing In Vitro and In Vivo

The AT3 in vivo gene silencing in mice was determined by singlesubcutaneous administration of the AT3 siRNA-ligand conjugate byfollowing a similar protocols described in Examples 33 for the TTR genesilencing in vivo. The in vitro gene silencing was evaluated byfollowing similar protocols described in Examples 31 and 32.

The siRNA conjugate 54944 and 56881 were subcutaneously administered toC57/BL6 mice at three different single dose levels: 5, 10 and 25 mg/kgand AT3 protein level relative to PBS control was measured 72 hour postdose. The results are shown in FIG. 2. Conjugates 54944 and 56881 arereferred to above and in FIG. 2 as 70001 and 70002, respectively.

Example 35: Synthesis of Mono-GalNAc Building Blocks for OligonucleotideConjugation

The mono-GalNAc building block shown can be prepared as shown in Scheme80.

Synthesis of 102: GalNAc acid 100 (8.39 g, 18.71 mmol) and hydroxyproline amine (10.00 g, 18.77 mmol) were taken together indichloromethane. HBTU (10.68 g, 28.12 mmol) and DIEA (9.80 mL, 3 eq.)were added and stirred the mixture for 2 his at ambient temperature. TheTLC was checked and the reaction mixture was transferred to a reparatoryfunnel and washed with water and brine. The organic layer was dried oversodium sulfate and the solvent removed. Crude product was purified bysilica gel chromatography using dichloromethane and MeOH as solvents toget the compound 102 as a pale yellow fluffy solid (11.77 g, 63%). NMR(400 MHz, DMSO) δ 7.80 (d, J=9.2 Hz, 1H), 7.69 (t, J=5.6 Hz, 1H),7.39-7.09 (m, 9H), 6.86 (ddd, J=9.0, 5.4, 2.1 Hz, 4H), 5.20 (d, J=3.4Hz, 1H), 5.03-4.83 (m, 2H), 4.47 (d, J=8.5 Hz, 1H), 4.41-4.07 (m, 2H),4.04-3.95 (m, 3H), 3.86 (dt, J=11.2, 8.9 Hz, 1H), 3.79-3.68 (m, 6H),3.68-3.36 (m, 3H), 3.21-2.88 (m, 5H), 2.26-2.14 (m, 2H), 2.09 (s, 3H),2.02 (t, J=6.7 Hz, 2H), 1.98 (s, 3H), 1.87 (d, J=7.5 Hz, 3H), 1.76 (s,3H), 1.53-1.29 (m, 7H).

Synthesis of 104: Hydroxy proline derivative 102 (6.00 g, 6.24 mmol) wasdissolved in dichloromethane (1.00 mL). To that DIEA (2.20 mL, 3 eq) andchloroamidite reagent were added. The reaction mixture was stirred for30 minutes and checked by TLC. It was transferred to a separatory funneland washed with water and sodium bicarbonate solution. The organic layerwas dried over sodium sulfate and the crude product was purified bysilica gel chromatography using dichloromethane and WON as eluent to getthe compound as a white fluffy solid. ¹H NMR (400 MHz, DMSO) δ 7.80 (d,J=9.2 Hz, 1H), 7.68 (s, 1H), 7.42-7.06 (m, 8H), 7.01-6.73 (m, 4H), 5.20(d, J=3.3 Hz, 1H), 4.96 (dd, J=11.2, 3.3 Hz, 1H), 4.63 (d, J=4.7 Hz,1H), 4.47 (d, J=8.5 Hz, 1H), 4.15 (s, 1H), 4.01 (s, 3H), 3.86 (d, J=11.0Hz, 1H), 3.70 (d, J=16.5 Hz, 9H), 3.45 (ddd, J=37.0, 23.3, 16.4 Hz, 6H),2.99 (dd, J=12.3, 6.4 Hz, 3H), 2.74 (dd, J=9.2, 5.8 Hz, 2H), 2.21 (s,2H), 2.09 (s, 3H), 2.05-1.95 (m, 5H), 1.88 (s, 3H), 1.76 (s, 3H),1.52-1.16 (m, 11H), 1.16-1.02 (m, 11H). ³¹P NMR δ=151.78, 151.61,151.50, 151.30.

Synthesis of 105: Compound 102 (2.10 g, 2.18 mmol) was dissolved in DCM(20 mL). To this mixture, succinic anhydride (0.441 g, 4.36 mmol) andDMAP (0.532 g, eq) followed by TEA (1 ml) were added. The reactionmixture was stirred overnight at room temperature. The TLC of thereaction mixture was checked and the reaction mixture was washed withwater and brine. The organic layer was dried over sodium sulfate and thecrude product filtered through a small pad of silica gel. Solvent wasremoved and this material was used for the next reaction. The succinatefrom the above reaction was dissolved in anhydrous acetonitrile. HBTU(1.59 g, 4.20 mmols) and DIEA (1.10 ml) were added and the mixture wasswirled for 5 minutes. A polystyrene solid support was added to thereaction mixture and the mixture was shaken overnight at ambienttemperature. The solid support was filtered, and washed and capped usingacetic anhydride/Py mixture. The solid support was again washed withdichloromethane. MeOH/DCM and ether (27.10 g, 55 umol/g).

Example 36: Synthesis of Mono-GalNAc NHS Esters for OligonucleotideConjugation

Mono-GalNAc NHS esters useful for oligonucleotide conjugation can beprepared as shown in Schemes 81-86 below.

Example 37: Synthesis of Amide- and Carbamate-Linked Building Blocks forOligonucleotide Conjugation

Synthesis of Compound 5001: To a stirred solution of compound 5000(23.22 g, 63.6 mmol) in DCM was added NaN₃ (12.4 g, 190.8 mmol) andTBAHS (21.6 g, 63.6 mmol) followed by the addition of 150 mL of asaturated NaHCO₃ solution. The resulting mixture was stirred for 14hours. The reaction mixture was then extracted with ethyl acetate (3×250ml), washed with water, brine and dried over anhydrous Na₂SO₄.Concentration of the solvent gave the crude material. This material wasdissolved in ethyl acetate (150 mL) followed by the addition of 150 mLof hexane, which resulted in precipitation of the product as a whitesolid. The solid was dried under reduced pressure to afford compound5001 (16.2 g, 68.4%). LCMS for compound 5001: Calculated: 372.33 (M⁺).Found: 407.1 (M⁻+Cl⁻).

Synthesis of compound 5002: To a stirred solution of compound 5001 (13.2g, 35.5 mmol) in THF (600 mL) was added PtO₂ (0.6 g) and the reactionmixture was stirred under hydrogen atmosphere at room temperature for 14hours. The catalyst was removed by filtration. Concentration of thesolvent afforded compound 5002 (13.0 g).

Synthesis of compound 5003: To a stirred solution of compound 5002 (10.0g, 28.89 mmol) and glutaric anhydride (3.29 g, 28.89 mmol) in DCM (100mL) were added pyridine (4.6 g) and DMAP (0.176 g). The reaction mixturewas stirred for 14 hours. The resulting mixture was concentrated, thensubjected to a filter column to afford compound 5003 (11.45 g, 86%).LCMS for compound 5003: Calculated: 460.43 (M⁺). Found: 459.3 (M⁻−1),495.0 (M⁻+Cl⁻).

Synthesis of compound 5004: To a stirred solution of compound 5003 (7.6g, 16.51 mmol). NHS (2.09 g, 18.16 mmol) and EDC (3.8 g, 19.8 mmol) inDCM (100 mL) was added DIEA (7.16 mL, 41.27 mmol) dropwise. Theresulting mixture was stirred for 14 hours. 100 mL of water was thenadded, and the product was extracted with DCM (2×50 mL), washed withcitric acid (20%), saturated NaHCO₃, brine, then dried over anhydrousNa₂SO₄. Concentration of the solvent afforded compound 5004 (6 g, 65%).LCMS for compound 5004: Calculated: 557.5 (M⁺). Found: 558.0 (M⁺+1).

Synthesis of compound 5005: To a stirred solution of compound 5002 (3.0g, 8.66 mmol) in acetonitrile (50 mL) was added DSC (2.22 g, 8.66 mmol)and the resulting mixture was stirred overnight (14 hours) at roomtemperature. The solvent was concentrated, then the product wasextracted with ethyl acetate (3×50 mL), washed with water, 10% citricacid, brine and dried over anhydrous Na₂SO₄. Concentration of thesolvent afford compound 5005 (3.8 g, 95%). LCMS Calculated forC₁₉H₂₅N₃O₁₂: 487.41 (M⁺). Found: 488.1 (M⁺+1), 510.1 (M⁺+Na⁺).

Synthesis of compound 5006: To a stirred solution of compound 5005(0.663 g, 1.36 mmol) in DCM (15 mL) were added amine (0.526 g, 1.5 mmol)and triethylamine (0.4 mL). The solvent was concentrated, then theproduct was extracted with ethyl acetate (3×50 mL), washed with water,10% citric acid, brine and dried over anhydrous Na₂SO₄. Concentration ofthe solvent afforded compound 5006 (0.7 g, 93%). LCMS Calculated forC₂₅H₃₃N₃O₁₁ 551.54 (M⁺). Found: 552.2 (M⁺+1), 574.2 (M⁺+Na⁺).

Synthesis of compound 5007: To a stirred solution of compound 5005 (0.7g, 1.26 mmol) in EtOH (15 was added Pd/C (0.1 g) and the resultingmixture was stirred under a hydrogen atmosphere overnight (14 h). Thecatalysts was removed by filtration over celite, and the mixture waswashed with EtOH (950 mL) and concentrated to afford the product whichwas used for the next step without purification. To a stirred solutionof the above acid in DCM (20 mL) were added EDC (488 mg, 2.56 mmol). NHS(730 mg, 6.35 mmol) and DIEA (0.88 mL, 5.07 mmol). The reaction mixturewas stirred overnight. Concentration of the reaction mixture followed bycolumn chromatography afforded compound 5007 (250 mg, 35%). LCMSCalculated for C₂₂H₃₀N₄O₁₃: 558.49 (M⁺). Found: 559.2 (M⁺+1), 581.1(M⁺+Na⁺).

Example 38: Synthesis of S- and C-Linked GalNAc Derivatives and theBuilding Blocks

Synthesis of compound 5012: Compound 4999 (15.6 g, 40.1 mmol) wastreated with TMSOTf (7.98 mL, 44.1 mmol) in DCE to afford compound 5011.Molecular weight for C₁₄H₂₀NO₈ (M+H)⁺ Calc. 330.12. Found 330.0.Compound 5011 (1.65 g, 5 mmol) and tert-butyl 5-mercaptopentanoate (1.0g, 5.25 mmol) in DCE were treated with TMSOTf (0.181 mL, 1.0 mmol)overnight. Aqueous work-up and silica gel column purification affordedcompound 5012 (380 mg, 0.731 mmol, 15%). Molecular weight forC₂₃H₃₇NNaO₁₀S (M+H)⁺ Calc 542.20. Found 542.1.

Synthesis of compound 5013: To a solution of compound 5012 (380 mg,0.731 mmol) in CH₂Cl₂ (4 mL) was added TFA (1 mL) at 0° C. The mixturewas stirred at 0° C. for 2 hours then at room temperature for 3 hours.The solvent was evaporated and the residue was co-evaporated withtoluene to afford crude compound 5013. This material was used for nextstep without purification. Molecular weight for C₁₉H₃₀NO₁₀S (M+H)⁺ Calc464.1590. Found 464.1.

Synthesis of compound 5014: Compound 5013 from the previous step (˜0.731mmol) was treated with N-hydroxy succinimide (168 mg, 1.46 mmol) in thepresence of EDCl (280 mg, 1.46 mmol) and DIEA (0.764 mL, 4.38 mmol) inCH₂Cl₂ (5 mL) for 14 hours. Aqueous work-up then column chromatographyafforded compound 5014 (284 mg, 0.507 mmol, 69% over 2 steps). Molecularweight for C₂₃H₃₃N₂O₁₂S (M+H)⁺ Calc. 561.1754. Found 561.1.

S-alkylation of compound 5009 with alkyne bromide affords compound 5010.

Compound 5016 is prepared from compound 5015 (see, J. Org. Chem., 67,2995-2999, 2002). The acid 5017 is prepared according to the reportedprocedure.

Synthesis of compound 5018: Compound 5017 (2.40 g, 5.18 mmol) wastreated with N-hydroxysuccinimide (716 mg, 6.22 mmol) in the presence ofEDCI (1.19 g, 6.22 mmol) and DIEA (2.70 mL, 15.5 mmol) in CH₂Cl₂, (30mL) for 14 h. Aqueous work-up followed by column chromatography affordedcompound 5018 (1.83 g, 3.26 mmol, 63%). Molecular weight forC₂₃H₃₃N₂O₁₂S (M+H)⁺ Calc. 561.1754. Found 561.2.

Compound 5024 was prepared using reported procedures (see, J. Org.Chem., 71, 3619-362, 2006 and Carbohydrate Research, 309, 319-330,1998).

Synthesis of compound 5025: Compound 5024 (1.94 g, 5.22 mmol), benzyl4-pentenoate (2.99 g, 15.7 mmol) and Grubbs Catalyst, 2nd Generation(433 mg, 0.522 mmol) in CH₂Cl₂ (20 mL) were heated at 40° C. for 40hours. The solvent was removed and the residue was purified by silicagel column chromatography to afford compound 5025 (1.87 g, 3.50 mmol,67%). Molecular weight for C₂₇H₃₆NO₁₀ (M+H)⁺ Calc. 534.2339. Found534.2.

Synthesis of compound 5028: To a solution of compound 5025 (1.85 g, 3.47mmol) in EtOAc (30 mL) was added palladium on carbon (Aldrich:330108-50G, 10 wt. %, Degussa type E101 NE/W: 185 mg). The reactionmixture was stirred under an atmosphere of hydrogen for 14 hours. Afterfiltration through Celite, the filtrate was removed in vacuo. Theresidue was purified by silica gel column chromatography to affordcompound 5028 (903 mg, 2.03 mmol, 59%). Molecular weight for C₂₀H₃₂NO₁₀(M+H)⁺ Calc. 446.2026. Found 446.1.

Synthesis of compound 5031: Compound 5028 (326 mg, 0.732 mmol) wastreated with N-hydroxysuccinimide (127 mg, 1.10 mmol) in the presence ofEDCI (211 mg, 1.10 mmol) and DIEA (0.383 mL, 2.20 mmol) in CH₂Cl₂ (5 mL)for 14 hours. Aqueous work-up followed by column chromatography affordedcompound 5031 (300 mg, 0.553 mmol, 76%). Molecular weight forC₂₄H₃₅N₂O₁₂ (M+H)⁺ Calc. 543.2190. Found 543.2.

Oxidative cleavage of compound 5024 affords aldehyde 5026. It is reducedto alcohol and then O-alkylation with benzyl-protected triflate followedby deprotection and esterification affords compound 5032. Reductiveamination of compound 5026 affords acid compound 5030 which isesterified to afford compound 5033.

Compound 5048 was prepared in a similar manner to that reported in theliterature (see, J. Org. Chem., 61, 6442-6445, 1996). Compounds 5046,5047, and 5048 are prepared in an analogous manner to that shown inScheme 91.

Example 39

Synthesis of compound 5050: N-Acetyl glucosamine (10 g) was refluxedwith hexenol (excess) in the presence of BF₃.Et₂O to afford compound5050.

Synthesis of compound 5052: Compound 5050 was treated with pivolylchloride as reported in the literature. This pivolyl ester was treatedwith triflic anhydride followed under reflux with water to affordcompound 5052.

Synthesis of compound 5055: Compound 5052 was first treated with sodiumhydroxide to remove pivolyl ester, then the resulting trihydroxylderivate was treated with benzoic anyhydride to afford compound 5054.The double bond was later oxidized to afford the carboxylic acid 5055.

Synthesis of compound 5056: Compound 5055 (502 mg, 0.732 mmol) wastreated with N-hydroxysuccinimide (127 mg, 1.10 mmol) in the presence ofEDCI (211 mg, 1.10 mmol) and DIEA (0.383 mL, 2.20 mmol) in CH₂Cl₂ (5 mL)for 14 hours. Aqueous work-up followed by column chromatography affordedcompound 5056 (400 mg, 0.553 mmol, 76%). Molecular weight forC₃₈H₃₈N₂O₁₃ (M+H)⁺ Calc. 730.24. Found 730.25.

Example 40: Synthesis of New GalNAc Conjugates Using Post-SyntheticMethods

The GalNAc groups in the table below were conjugated to an siRNA by theprocedures described in Schemes 94, 95 or 96, above.

GalNAc NHS Esters (RCOO-NHS) R

Example 41: siRNA-Ligand Conjugates

siRNA-Ligand conjugates were prepared. The siRNA in each conjugate wasthe same and targeted to TTR. The following ligands were attached to the3′ end of the sense strand of each siRNA. The left side of the liganddescription indicates the site of attachment to the 3′ terminus of thesense strand.

TTR siRNA Ligand Conjugates siRNA Ligand (attached to 3′ terminusConjugate of sense strand) 43527 L96 60148 Q150Q150L193 60146(T3gs)(T3gs)(T3g) 60142 (Tgs)(Tgs)(Tg) 60133 Q155Q155L199 60139Q160Q160L204 60134 Q156Q156L200 60132 Q154Q154L198 60125 Q161Q161L20760124 Q159Q159L203 60122 L203 60123 L206 60135 L207 60136 L208 60129L198 60126 L197 60131 L200 60127 L202 60130 L199 60128 L201 60137 L20460138 L205 60140 (Tg)(Tg)(Tg) 60144 (T3g)(T3g)(T3g) 60141 (Tg) 60143s(Tg) 60145 (T3g) 60147 s(T3g)

The siRNA-Ligand conjugates 43527, 60148, 60146, 60142, 60133, 60139,60134, 60132 and/or 60125 are tested in mice as described in Example 33.The results are provided in FIG. 1.

Example 42: siRNA-Ligand Conjugates

siRNA-Ligand conjugates were prepared. The siRNA in each conjugate wastargeted to TTR. The following ligands were attached to the sense strandof the siRNA at the positions indicated.

TTR siRNA Ligand Conjugates siRNA Ligand (5′-3′) 56718 -(Uyg)(Ayg)(Ayg)56719 -(Cyg)(Uyg)(Ayg)- (These are the 4^(th), 5^(th) and 6^(th)nucleotides from the 3′ end of the siRNA) 56720 -(Gyg)(Cyg)(Uyg)- (Theseare the 7^(th), 8^(th) and 9^(th) nucleotides from the 3′ end of thesiRNA) 56721 -(Cyg)(Uyg)(Uyg)- (These are the 10^(th), 11^(th) and12^(th) nucleotides from the 3′ end of the siRNA) 56722-(Gyg)(Uyg)(Uyg)- (These are the 7^(th), 8^(th) and 9^(th) nucleotidesfrom the 5′ end of the siRNA) 56723 -(Ayg)(Gyg)(Uyg)- (These are the4^(th), 5^(th) and 6^(th) nucleotides from the 5′ end of the siRNA)56724 (Ayg)(Ayg)(Cyg)- (These are at the 5′ end of the siRNA) 56725(Ayg) This ligand is at each of the 5′ and 3′ ends of the siRNA (Uyg)This ligand is the 10^(th) nucleotide from the 3′ end of the siRNA 56726(Ayg) This ligand is at each of the 5′ and 3′ ends of the siRNA (Uyg)This ligand is the 11^(th) nucleotide from the 3′ end of the siRNA 56727(Ayg) This ligand is at the 3′ end and is also the 4^(th) nucleotidefrom the end (Uyg) This ligand is the 7^(th) nucleotide from the 3′ end56729 (Uyg)(Uyg)(Tyg) (These ligands are at the 3′ end of the siRNA)55727 L96 (This ligand is at the 3′ end of the siRNA)

The Chemical Structures for Uyg, Ayg, Gyg and Cyg are Given in the Tableof Chemical Groups Provided at the End of the Experimental Section.

FIGS. 4-7 show the binding affinities of the siRNA-ligand conjugates56718-56727, 56729 and 55727.

Example 43: siRNA-Ligand Conjugates

The siRNA-Ligand conjugates in the table below were prepared. The siRNAin each conjugate was the same and targeted to AT3. The followingligands were attached to the sense strand of each siRNA as indicated.

AT3 siRNA Ligand Conjugates siRNA Sequence 5′-3′ 56874 Q151L96 (at the3′ end of the siRNA) 56875 Q151Q151L96 (at the 3′ end of the siRNA)56876 Q151 (at the 5′ end of the siRNA) L96 (at the 3′ end of the siRNA)56877 Q151 (at the 5′ end of the siRNA) 56878 Q150L96 (at the 3′ end ofthe siRNA) 56879 Q150Q150L96 (at the 3′ end of the siRNA) 56880Q150Q150Q150L96 (at the 3′ end of the siRNA) 56881 Q150Q150L193 (at the3′ end of the siRNA) 56882 L193 (at the 3′ end of the siRNA) 54944 L96(at the 3′ end of the siRNA)

Example 44

The siRNA-Ligand conjugates in the table were prepared. The siRNA ineach conjugate was the same and targeted to AT3. The ligands wereattached to the sense strand at the positions indicated in the table.

FIGS. 8 and 9 show the binding affinities of the SIRNA-ligand conjugates56876, 66875, 56874, 66878, 56880, 56879, 54944, 56877, 56881 and/or56882. The Ki values for these conjugates are reported below.

Sense Sequence Duplex (5′ to 3′) # GalNac/position Ki(nM) 56876 Q151 (atthe 5′ end 6 (2 tri), 3′ & 5.168 of the siRNA) 5′ end L96 (at the 3′ endof the siRNA) 56875 Q151Q151L96 (at the 3′ 9 (3 tri), 6.372 end of thesiRNA) 3′ end 56874 Q151L96 (at the 3′ 6 (2 tri), 7.174 end of thesiRNA) 3′ end 56878 Q150L96 (at the 3′ 4 (1 + 1 tri), 11.72 end of thesiRNA) 3′ end 56880 Q150Q150Q150L96 (at the 6 (1 + 1 + 1 + 1 tri), 11.933′ end of the siRNA) 3′ end 56879 Q150Q150L96 (at the 3′ 5 (1 + 1 + 1tri), 15.66 end of the siRNA) 3′ end 54944 L96 (at the 3′ 3 (1 tri),19.085 end of the siRNA) 3′ end 56877 Q151 (at the 5′ 3 (tri), 20.39 endof the siRNA) 5′ end 56881 Q150Q150L193 (at the 3′ 3 (1 + 1 + 1), 51.34end of the siRNA) 3′ end 56882 L193 (at the 3′ 1, 3′ end N/A end of thesiRNA)

Example 45: siRNA's with Different GalNAc Ligands (TriantennaryDerivatives)

siRNA-Ligand conjugates were prepared. The siRNA in each conjugate wasthe same and targeted to TTR. The following ligands were attached to the3′ end of the sense strand of each siRNA. The left side of the liganddescription indicates the site of attachment to the 3′ terminus of thesense strand.

TTR siRNA Ligand Conjugates siRNA Ligand (attached to 3′ terminusConjugate of sense strand) 60123 L206 -- Urea, Trianternnary(3) 60136L208 -- Alpha, Trianternnary(3) 60126 L197 -- Amide, Trianternnary(3)60127 L202 -- Beta-Thio, Trianternnary(3) 60128 L201 -- Alpha-Thio,Trianternnary(3) 60138 L205 -- Alpha, C-glycoside, Trianternnary(3)43527 L96 -- , Trianternnary (Control)

FIG. 10 shows the in vivo efficacy of the triantennary GalNAc ligands43527, 60126, 60138, 60128, 60127, 60316, and 60123 (at 15 mg/kg and 5mg/kg doses) after 72 and 144 hours.

Example 46: siRNA's with Different GalNAc Ligands (TriantennaryDerivatives (1+1+1))

siRNA-Ligand conjugates were prepared. The siRNA in each conjugate wasthe same and targeted to TTR. The following ligands were attached to the3′ end of the sense strand of each siRNA. The left side of the liganddescription indicates the site of attachment to the 3′ terminus of thesense strand.

TTR siRNA Ligand Conjugates siRNA Ligand (attached to 3′ terminusConjugate of sense strand) 60124 Q159Q159L203 -- Urea, 1 + 1 + 1 60125Q161Q161L207 -- Alpha, 1 + 1 + 1 60132 Q154Q154L198 -- Amide, 1 + 1 + 160134 Q156Q156L200 -- Beta-Thio, 1 + 1 + 1 60133 Q155Q155L199 --Alpha-Thio, 1 + 1 + 1 60139 Q160Q160L204 -- Alpha, C-glycoside, 1 + 1 +1 60148 Q150Q150L193 -- Beta, 1 + 1 + 1siRNA conjugates directed to AT3, two of which are described in Examples30 and 34, were prepared and tested. The structure of 58137 is providedbelow. Each conjugate had the same AT3 siRNA sequence. FIG. 11 shows thein vivo efficacy of triantennary GalNAc ligands 54944, 56881 and 58137[(1+1+1) design].

siRNA Ligand (attached to 3′ terminus Conjugate of sense strand) 54944L96 56881 Q150Q150L193 58137 (Uyg)(Uyg)(Uyg)

siRNA conjugates directed to TTR were prepared and tested. Eachconjugate had the same TTR siRNA sequence. The following ligands wereattached to the 3′ end of the sense strand of each siRNA. The left sideof the ligand description indicates the site of attachment to the 3′terminus of the sense strand. FIG. 12 shows the in vivo efficacy oftriantennary GalNAc ligands 55727, 58138 and 58139 [(1+1+1) design]. Theligand designs for duplexes 55727, 58138 and 58139 are provided below,

siRNA Ligand (attached to 3′ terminus of Conjugate sense strand) 55727L96 58138 Q150Q150L193 58139 (Uyg)(Uyg)(Uyg)

Example 47

Synthesis of compound 102: GalNAc acid 100 (8.39 g, 18.71 mmol) andhydroxy proline amine (10.00 g, 18.77 mmol) were taken together indichloromethane. HBTU (10.68 g, 28.12 mmol) and DIEA (9.80 mL, 3 eq.)were added and the mixture was stirred for 2 hours at ambienttemperature. The product was checked by thin layer chromatography andthe reaction mixture was transferred to a separatory funnel and washedwith water and brine. The organic layer was dried over sodium sulfateand the solvent was removed. The crude product was purified by silicagel chromatography using dichloromethane and MeOH as solvents to affordcompound 102 as a pale yellow fluffy solid (11.77 g, 63%). ¹H NMR (400MHz, DMSO-d₆): δ 7.80 (d, 0.1=9.2 Hz, 1H), 7.69 (t, J=5.6 Hz, 1H),7.39-7.09 (m, 9H), 6.86 (ddd, J=9.0, 5.4, 2.1 Hz, 4H), 5.20 (d, J=3.4Hz, 1H), 5.03-4.83 (m, 2H), 4.47 (d, J=8.5 Hz, 1H), 4.41-4.07 (m, 2H),4.04-3.95 (m, 3H), 3.86 (dt, J=11.2, 8.9 Hz, 1H), 3.79-3.68 (m, 6H),3.68-3.36 (m, 3H), 3.21-2.88 (m, 5H), 2.26-2.14 (m, 2H), 2.09 (s, 3H),2.02 (t, J=6.7 Hz, 2H), 1.98 (s, 3H), 1.87 (d, J=7.5 Hz, 3H), 1.76 (s,3H), 1.53-1.29 (m, 7H).

Synthesis of compound 104: Hydroxy proline derivative 102 (6.00 g, 6.24mmol) was dissolved in dichloromethane (100 mL). DIEA (2.20 mL, 3 eq)and 2-cyanoethyl diisopropylchlorophosphoramidite were added. Thereaction mixture was stirred for 30 minutes and checked by thin layerchromatography. The mixture was transferred to a separatory funnel andwashed with water and sodium bicarbonate solution. The organic layer wasdried over sodium sulfate and the crude product was purified by silicagel chromatography using dichloromethane and MeOH as eluent to affordthe compound as a white fluffy solid. ¹H NMR (400 MHz, DMSO-d₆): δ 7.80(d, J=9.2 Hz, 1H), 7.68 (s, 1H), 7.42-7.06 (m, 8H), 7.01-6.73 (m, 4H),5.20 (d, J=3.3 Hz, 1H), 4.96 (dd, J=11.2, 3.3 Hz, 1H), 4.63 (d, J=4.7Hz, 1H), 4.47 (d, J=8.5 Hz, 1H), 4.15 (s, 1H), 4.01 (s, 3H), 3.86 (d,J=11.0 Hz, 1H), 3.70 (d, J=16.5 Hz, 9H), 3.45 (ddd, J=37.0, 23.3, 16.4Hz, 6H), 2.99 (dd, J=12.3, 6.4 Hz, 3H), 2.74 (dd, 0.1=9.2, 5.8 Hz, 2H),2.21 (s, 2H), 2.09 (s, 3H), 2.05-1.95 (m, 5H), 1.88 (s, 3H), 1.76 (s,3H), 1.52-1.16 (m, 11H), 1.16-1.02 (m, 11H). ³¹P NMR: δ 151.78, 151.61,151.50, 151.30.

Synthesis of compound 105: Compound 102 (2.10 g, 2.18 mmol) wasdissolved in DCM (20 mL). To this mixture succinic anhydride (0.441 g,4.36 mmol) and DMAP (0.532 g, eq) followed by TEA (1 ML) were added. Thereaction mixture was stirred overnight at room temperature. The reactionwas checked by thin layer chromatography, then the reaction mixture waswashed with water and brine. The organic layer was dried over sodiumsulfate and the crude product filtered through a small pad of silicagel. The solvent was removed and this material was used for the nextreaction. The succinate from the above reaction was dissolved inanhydrous acetonitrile. HBTU (1.59 g, 4.20 mmols) and DEA (1.10 ml) wereadded and the mixture was swirled for 5 minutes. A polystyrene solidsupport was added to the reaction mixture and the mixture was shakenovernight at ambient temperature. The solid support was filtered, washedand capped using acetic anhydride/pyridine mixture. The solid supportwas again washed with dichloromethane. MeOH/DCM and ether (27.10 g, 55μmol/g).

Example 48: Synthesis of Amino Linkers for Post-Synthetic Conjugation

Compound 101A: Z-Aminocaproic acid (22.2 g, 82.50 mmol) was dissolved inDMF (250 mL) and cooled to 0° C. To the solution were added diisopropylethyl amine (44.4 mL, 275 mmols), HBTU (40.4 g, 106.7 mmol), and HOBT(30.0 g, 220 mmol). After stirring under argon for 20 minutes at 0° C.,4-Hydroxy-L-proline methyl ester hydrochloride (20.0 g, 110 mmol) wasadded and the stirring was continued under argon at room temperatureovernight. The reaction mixture was evaporated to dryness. To theresidue ethyl acetate (250 mL) was added. The organic layer was washedwith water, saturated sodium bicarbonate, water again, and saturatedsodium chloride. The organic layer was dried over sodium sulfate,filtered and evaporated to dryness. Crude compound 101A (Rf=0.5 in 10%MeOH/DCM, 24.30 g) was obtained. Compound 101A was purified by columnchromatography first by eluting with 2% methanol/dichloromethane toremove impurities followed by 5% methanol/dichloromethane to afford21.36 g (65%) of product. 1H NMR (400 MHz, DMSO-d₆): δ 7.35 (m, 5H),5.15 (d, OH, D₂O exchangeable), 4.99 (s, 2H), 4.27 (m, 1H), 3.97 (m,1H), 3.58 (s, 1H) 3.20-3.47 (m, 5H), 2.94-3.02 (m, 2H), 2.10-2.32 (m,2H), 1.74-2.01 (m, 2H), 1.35-1.4 (m, 4H), 1.22-1.28 (m, 4H)

Compound 102A: Compound 101A (21.36 g, 54.43 mmol) was dissolved in THF(200 mL). The reaction mixture stirred under argon for 20 minutes at 0°C. Then lithium borohydride (1.19 g, 54.43 mmol) was added to thesolution over 20 minutes at 0° C., and the stirring was continued underargon at room temperature overnight. The reaction mixture was cooled to0° C. The excess lithium borohydride was quenched with 5M NaOH (30 mL).After stirring for 30 minutes the reaction mixture was evaporated todryness. To the residue dichloromethane (200 mL) was added. The organiclayer was washed with water and saturated sodium chloride. The organiclayer was dried over sodium sulfate, filtered and evaporated to dryness.Crude compound 102A (Rf=0.4 in 10% MeOH/DCM, 35.88 g) was obtained.Compound 102A was purified by column chromatography by eluting with 3%methanol/dichloromethane to remove impurities followed by 5%methanol/dichloromethane to afford 9.21 g (49%) of product. ¹H NMR (400MHz, DMSO-d₆): δ 7.35 (m, 5H), 4.99 (s, 2H), 4.91 (d, OH, D₂Oexchangeable), 4.77 (t, OH, D₂O exchangeable), 4.27 (m, 1H), 3.97 (m,1H), 3.20-3.47 (m, 5H), 2.94-3.02 (m, 2H), 2.10-2.32 (m, 2H), 1.74-2.01(m, 2H), 1.35-1.4 (m, 4H), 1.22-1.28 (m, 4H).

Compound 103A: Compound 102A (9.21 g, 25.27 mmols) was co-evaporatedwith anhydrous pyridine (80 mL) twice. Then the compound was placedunder hard vacuum overnight to dry. Compound 102A was taken from hardvacuum and dissolved in anhydrous pyridine (200 mL). To this solution acatalytic amount of dimethylamino pyridine (0.35 g, 2.53 mmol) wasadded. The reaction mixture stirred under argon for 30 minutes at 0° C.,Then DMT-Cl (9.0 g, 26.53 mmols) was added to the solution at 0° C. Themixture was stirred under vacuum followed by argon, and stirring wascontinued under argon at room temperature overnight. The excess DMT-Clwas quenched by the addition of methanol (15 mL). The reaction mixturewas evaporated to dryness, and to the residue dichloromethane (200 mL)was added. The organic layer was washed with water, saturated sodiumbicarbonate, water again, and saturated sodium chloride. The organiclayer was dried over sodium sulfate, filtered and evaporated to dryness.Crude compound 103A (Rf=0.6 in 100% EtOAc, 14.02 g) was obtained.Compound 103A was purified by column chromatography by first elutingwith 50% ethyl acetate (1% TEA) in hexanes to remove impurities followedby 100% ethyl acetate (1% TEA) to afford 12.36 g (73.4%) of the productas a white foamy solid. ¹H NMR (400 Wiz, DMSO-d₆): δ 7.17-7.33 (m, 14H),4.99 (s, 2H), 4.91 (d, OH, D₂O exchangeable), 4.37 (m, 1H), 4.01 (m,1H), 3.72 (s, 6H) 3.56 (m, 1H) 3.29 (m, 1H), 3.14 (m, 1H), 2.93-3.02 (m,4H), 2.18 (m, 2H) 1.74-2.01 (m, 2H), 1.37-1.41 (m, 6H).

Compound 104A: Compound 103A (12.36 g, 18.54 mmol) was dissolved in 10%methanol/ethyl acetate (300 mL) and purged with argon. To the reactionmixture was added 10% palladium by wt. on active carbon wet Degussa type(1.3 g). The flask was re-purged with argon. The flask was purged withhydrogen twice, and then hydrogen was bubbled through the reactionmixture for 10 seconds. The reaction mixture continued to stir underhydrogen at room temperature overnight. The reaction mixture wasdecanted onto a sintered funnel packed with celite and washed twice withmethanol. The organic layer was evaporated to dryness affording compound104A (eluent 10% MeOH in DCM, 9.16 g, 93%) as a white solid, whichrequired no further purification. ¹H NMR (400 MHz, DMSO-d₆): δ 7.15-7.31(m, 9H), 6.86 (m, 4H) 4.99 (s, 1H), 4.37 (m, 1H), 4.01 (m, 2H), 3.72 (s,6H) 3.56 (m, 1H) 3.29 (m, 1H), 3.14 (m, 1H), 2.93-3.02 (m, 2H), 2.45 (m,2H), 2.18 (m, 2H) 1.74-2.01 (m, 2H), 1.37-1.41 (m, 3H) 1.13-1.38 (m,4H). MS: 533.4 (+H), 555.3 (+Na).

Compound 105A: Compound 104A (9.16 g, 17.2 mmol) was dissolved indichloromethane (200 mL). The reaction mixture stirred under argon for10 minutes at 10° C. To the reaction mixture triethylamine (4.80 mL,34.4 mmol) was added drop wise as the mixture continued to stir underargon for 20 minutes at 10° C. To the reaction mixture ethyltrifluoroacetate (3.05 mL, 25.8 mmol) was added drop wise as the mixturecontinued to stir under argon for 10 minutes at 10° C. The reactionmixture continued to stir under argon at room temperature overnight. Thereaction mixture was washed with water and saturated sodium chloride.The organic layer was dried over sodium sulfate, filtered and evaporatedto dryness. Crude Compound 105A (Rf=0.6 10% MeOH in DCM, 10.89 g) wasobtained. Upon column purification by eluting with 5%methanol/dichloromethane (1% TEA) Compound 105A (8.76 g, 81%) wasobtained as a yellow foamy solid. ¹H NMR (400 MHz, DMSO-d₆): δ 7.56-7.09(m, 9H), 7.01-6.52 (m, 4H), 5.34-5.04 (m, 1H), 4.99-4.78 (m, 1H),4.48-4.25 (m, 2H), 3.83-3.67 (n, 6H), 3.60-3.50 (m, 1H), 3.49-3.18 (m,2H), 3.16-2.91 (m, 2H), 2.89-2.56 (m, 2H), 2.54-2.32 (m, 2H), 2.32-1.69(1n, 3H), 1.59-1.03 (m, 4H). ¹⁹F NMR (DMSO-d₆): −77.14 (s, 3F). MS:627.3 (−H), 663.3 (+Cl).

Compound 106A: Compound 105A (8.76 g, 13.93 mmol), DMAP (5.10 g, 41.79mmol), and triethylamine (3.90 mL, 27.86 mmol) were dissolved indichloromethane (300 mL). The reaction mixture was stirred under argonfor 10 minutes. Then, succinic anhydride (2.80 g, 27.86 mmol) was addedand the mixture continued stirring under argon at room temperatureovernight. The reaction mixture was washed with saturated sodiumchloride solution twice. The organic layer was dried over sodiumsulfate, filtered and evaporated to dryness. Compound 106A (Rf=0.9 10%MeOH in DCM, 10.87 g, 94%) was obtained as a white solid, which requiredno further purification. MS: 727.2 (−H), 763.2 (+Cl). The succinate thusobtained (2.00 g, 2.41 mmol) was dissolved in acetonitrile (100 mL). Tothe solution diisopropylethylamine (1.68 mL, 9.64 mmol) and HBTU (1.83g, 4.82 mmols) were added. The reaction mixture was shaken for 10minutes. Long chain amino alkyl controlled pore glass support (CPG) (27g) was added to the flask and the mixture continued to shake overnight.The CPG compound and reaction mixture were decanted over a sinteredfunnel. The reaction mixture was washed with 1%triethylamine/dichloromethane, followed by two washes of 10% methanol indichloromethane, another wash of 1% triethylamine in dichloromethane,and anhydrous diethyl ether. The CPG compound was suction dried for 1hour, then recovered from the funnel and placed under hard vacuum for 2hours. The CPG was capped with 25% acetic anhydride in pyridine (100 mL)and the mixture shook for 4 hrs. The washing procedure repeated as aboveand dried the compound under vacuum to obtain the CPG 106A (28 g, 67μmol/g).

Compound 107A: Compound 105A (8.89 g, 14.14 mmol) anddiisopropylethylamine (4.93 mL, 28.28 mmol) were dissolved in anhydrousdichloromethane (60 mL). The reaction mixture was stirred under argonfor 5 minutes. Then 2-cyanoethyl diisopropylchlorophosphoramidite (5.63mL, 16.26 mmol) was added to the reaction mixture. The reaction mixturecontinued stirring under argon at room temperature for 30 minutes. Thereaction mixture was diluted with dichloromethane (100 mL). The organiclayer was washed with water, saturated sodium bicarbonate, water again,and saturated sodium chloride. The organic layer was dried over sodiumsulfate, filtered and evaporated to dryness affording crude Compound107A (Rf=0.44 5% MeOH in DCM, 11.02 g). Upon column purification byeluting with 3% methanol in DCM (1% TEA). Compound 107A (6.31 g, 54%)was obtained as a yellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.37 (s,1H), 7.63 (d 1H), 7.42-6.98 (m, 8H), 6.92-6.77 (m, 4H), 4.25-3.90 (m,2H), 3.78-3.64 (m, 7H), 3.48 (d, 3H), 3.29 (d, 1H), 3.23-2.92 (m, 4H),2.86 (d, 1H), 2.73 (t, 1H), 2.58 (t, 1H), 2.53-2.47 (m, 4H), 2.33-1.87(m, 4H), 1.55-0.97 (m, 12H), ³¹P (DMSO-d₆): 151.68 (d, 1P).

Compound 118: Serinol 116 (2.05 g, 22.5 mmol) and compound 117 (8.03 g,24.75 mmol) were dissolved in DMF (120 mL) under argon. Triethylamine(5.0 mL, 67.5 mmol) was added to the reaction mixture, which was stirredat room temperature overnight. The reaction mixture was evaporated todryness and the residue was dissolved in ethyl acetate (120 mL). It wasthen washed with water (30 mL) and saturated sodium chloride (2×50 mL).The organic layer was dried over sodium sulfate, filtered and evaporatedto dryness. The crude compound (5.33 g, Rf=0.21 15% MeOH in DCM,) waspurified silica gel column chromatography using 10% methanol indichloromethane as eluent to afford compound 118 (4.98 g, 78%) as awhite foam. m/z: 324.13 (+Na). 1H NMR (400 MHz, DMSO-d₆): δ 9.39 (s, 2NH, D₂O exchangeable), 3.34-3.06 (m, 7H), 3.06-2.91 (m, 2 OH, D₂Oexchangeable), 2.30-2.00 (m, 2H), 1.97-0.86 (m, 6H), ¹³C NMR (101 MHz,CD₃CN): δ 172.18 (s), 156.41 (s), 117.50 (s), 60.30 (s), 52.85 (s),38.88 (s), 35.35 (s), 28.11 (s), 25.90 (s), 25.29 (s).

Compound 119: Compound 118 (3.70 g, 12.3 mmol) was co-evaporated withanhydrous pyridine (30 mL) twice, then dried under high vacuumovernight. It was then dissolved in anhydrous pyridine (90 mL). To thissolution a catalytic amount of DMAP (0.15 g, 1.23 mmol) was added andstirred the mixture under Argon for 30 minutes at 0° C. DMTr-Cl (4.38 g,12.9 mmol) was added to the solution at 0° C. and continued the stirringat room temperature for 2 hours. The volatiles were then removed undervacuum. The residue was dissolved in dichloromethane (150 mL) and washedwith water (2×100 mL) followed by saturated sodium chloride (100 mL).The organic layer was dried over sodium sulfate, filtered and evaporatedto dryness. The crude compound (6.83 g, Rf=0.44 in 5% MeOH in DCM) waspurified by silica gel column chromatography by first eluting withdichloromethane (with 1% TEA) followed by 3% methanol in dichloromethane(with 1% TEA) to obtain compound 119 (2.32 g, 23%) as a white foam. m/z:601.2 (−1), 637.2 (+Cl). ¹H NMR (400 MHz, DMSO-d₆): δ 9.38 (s, 2 NH, D₂Oexchangeable), 7.31-7.13 (m, 7H), 6.86 (d, J=8.9 Hz, 6H), 4.60 (t, J=5.2Hz, 1H), 3.72 (s, 6H), 3.54-3.23 (m, 6H), 2.95 (ddd, J=36.9, 8.8, 5.8Hz, 1 OH, D₂O exchangeable), 2.08 (t, J=7.4 Hz, 2H), 1.47 (tt, J=14.6,7.4 Hz, 2H), 1.34-1.12 (m, 4H). ¹³C NMR (101 MHz, CD₃CN): δ 172.83 (s),158.97 (s), 157.28 (s), 156.93 (s), 146.13 (s), 136.82 (s), 136.52 (s),130.70 (s), 128.68 (d, J=4.0 Hz), 127.49 (s), 118.41 (s), 115.54 (s),114.02 (s), 86.08 (s), 63.66 (s), 61.84 (s), 55.93 (s), 51.77 (s), 46.68(s), 40.99 (d, J=21.0 Hz), 40.72 (s), 40.68 (s), 40.47 (s), 40.26 (s),40.05 (s), 39.84 (s), 36.31 (s), 29.04 (s), 26.85 (s), 25.95 (s).

Compound 120: Compound 119 (1.0 g, 1.7 mmol), DMAP (620 mg, 5.1 mmol),and triethylamine (0.5 mL, 3.4 mmol) were dissolved in dichloromethane(15 mL). The reaction mixture was stirred under argon for 5 minutes.Then, succinic anhydride (340 mg, 3.4 mmol) was added and continuedstirring under argon at room temperature overnight. The reaction mixturewas diluted in dichloromethane (100 mL) washed with saturated sodiumchloride (2×25 mL). The organic layer was dried over sodium sulfate,filtered and evaporated to dryness. The crude compound (Rf=0.3 50% EtOAcin Hex) 1.01 g (98%) was obtained as a white solid, which was used forthe next reaction without further purification. MS: 702.3 (−H), 737.5(+Cl). ¹H NMR (400 MHz, DMSO-d₆): δ 9.42 (t, J=5.2 Hz, 2 NH, D₂Oexchangeable), 7.87 (s, 1 OH, D₂O exchangeable), 7.47-7.15 (m, 7H),6.99-6.74 (m, 6H), 4.32-3.94 (m, 3H), 3.90-3.42 (m, 8H), 3.31-2.72 (m,6H), 2.57-2.29 (m, 2H), 1.30-0.87 (m, 6H).

Compound 121: The succinate 120 (1.0 g, 1.4 mmol) was dissolved inacetonitrile (60 mL). To this solution diisopropylethylamine (1.15 mL,5.6 mmol) and HBTU (1.26 g, 2.8 mmol) were added. The reaction mixturewas swirled until all contents were dissolved. CPG (14 g) was added tothe flask and the mixture shook overnight. The CPG was filtered andwashed consecutively with dichloromethane, 10% methanol indichloromethane, dichloromethane and anhydrous diethyl ether. The CPGwas suction dried for 1 hour, then recovered from the funnel and placedunder hard vacuum for 2 hours. The loaded CPG was capped with 25% aceticanhydride/pyridine (100 mL) for three hours. The CPG was filtered andthe same washing procedure described earlier was repealed. The CPG wassuction dried for 1 hour and dried under high vacuum overnight to affordcompound 120 (14 g, 77 μmol/g).

Compound 122: L-Threoninol (3.54 g, 33.7 mmol) and compound 117 (12.0 g,37.1 mmol) were dissolved in DMF (90 mL) under argon. Triethylamine(14.0 mL, 101.1 mmol) was added to the reaction mixture, which stirredat room temperature overnight. The reaction mixture was evaporated todryness and the residue dissolved in ethyl acetate (120 mL). It was thenwashed with water (50 mL) and saturated sodium chloride (2×50 mL). Theorganic layer was dried over sodium sulfate, filtered and evaporated todryness. The crude compound (3.60 g, Rf=0.43 50% EtOAc in Hexane) waspurified silica gel column chromatography using 50% ethyl acetate inhexane followed by 100% ethyl acetate as eluent to afford compound 122(3.20 g, 87%) as a yellow foam. m/z: 315.1 (+H). ¹H NMR (400 MHz,DMSO-d₆): δ 8.76 (t, 2 NH, D₂O exchangeable) 3.93-3.21 (m, 1H),3.21-3.01 (m, 2 OH, D₂O exchangeable), 3.05-2.83 (m, 3H), 2.75-2.51 (m,1H), 2.55-2.37 (m, 1H), 2.20-1.98 (m, 2H), 1.70-1.39 (m, 7H), 1.28-1.06(m, 3H), ¹³C NMR (101 MHz, DMSO-d₆): δ 176.1 (s), 156.3 (s), 158.33 (s),65.36 (s), 64.20 (s), 60.51, 38.88 (s), 36.34 (s), 27.98 (s), 25.79 (s),25.70-24.33 (m), 20.05 (s).

Compound 123: Compound 122 (3.00 g, 9.60 mmol) was co-evaporated withanhydrous pyridine (15 mL) twice and dried under high vacuum overnight.It was then dissolved in anhydrous pyridine (90 mL). To this solution acatalytic amount of DMAP (0.12 g, 0.96 mmol) was added and the mixturewas stirred under argon for 30 minutes at 0° C. DMTr-Cl (3.39 g, 10.08mmol) was added to the solution at C and stirring was continued at roomtemperature for 2 hours. The volatiles were then removed under vacuum.The residue was dissolved in dichloromethane (150 mL) and washed withwater (2×100 mL) followed by saturated sodium chloride (100 mL). Theorganic layer was dried over sodium sulfate, filtered and evaporated todryness. The crude compound (7.02 g, Rf=0.6 in 25% EtOAc in Hexane) waspurified by silica gel column chromatography by first eluting with 5%ethyl acetate in hexane (with 1% TEA) followed by 15% ethyl acetate inhexane (with 1% TEA) to obtain compound 123 (700 mg, 11%) as a whitefoam. m/z: 615.2 (−1), 651.2 (+Cl). ¹H NMR (400 MHz, DMSO-d₆): δ 8.76(t, 2 NH. D₂O exchangeable) 7.87-6.93 (m, 7H), 6.93-6.63 (m, 6H),4.19-3.63 (m, 10H), 3.61 (m, 1OH, D₂O exchangeable), 2.74-1.98 (m, 4H),1.71-1.10 (m, 6H), 1.08-0.76 (m, 3H). ¹³C NMR (101 MHz, DMSO-d₆): δ172.12 (d, J=9.4 Hz), 157.88 (d, J=15.4 Hz), 145.13 (s), 140.22 (s),135.86 (d, J=6.4 Hz), 131.07-130.03 (m), 130.03-129.28 (m), 128.90 (s),127.66 (d, J=7.1 Hz), 127.38 (s), 126.45 (d, J=9.7 Hz), 112.89 (d,J=30.1 Hz), 85.08 (s), 65.00 (s), 63.22 (d, J=48.5 Hz), 54.97 (s), 53.72(s), 45.56 (s), 40.02 (d, J=21.0 Hz), 39.85-39.80 (m), 39.71 (s), 39.50(s), 39.29 (s), 39.08 (s), 38.88 (s), 35.24 (s), 28.02 (s), 25.86 (s),25.13 (d, J=13.6 Hz), 21.15 (s), 20.20 (s), 10.35 (s).

Compound 124: Compound 123 (600 mg, 1.0 mmol), DMAP (420 mg, 3.0 mmol),and triethylamine (0.4 mL, 2.0 mmol) were dissolved in dichloromethane(15 mL). The reaction mixture was stirred under argon for 5 minutes.Then succinic anhydride (230 mg, 2.0 mmol) was added and stirring wascontinued under argon at room temperature overnight. The reactionmixture was diluted in dichloromethane (50 mL) washed with saturatedsodium chloride (2×25 mL). The organic layer was dried over sodiumsulfate, filtered and evaporated to dryness. Compound 124 (Rf=0.6 25%EtOAc in hexane) 760 mg (99%) was obtained as a grey foam, which wasused for the next reaction without further purification. MS: 715.3 (−H).

Compound 125: Compound 124 (760 mg, 1.0 mmol) was dissolved inacetonitrile (60 mL). To this solution diisopropylethylamine (0.74 mL,4.0 mmol) and HBTU (800 mg, 2.0 mmol) were added. The reaction mixturewas swirled until all contents were dissolved. CPG (10 g) was added tothe flask and the mixture was shaken overnight. The CPG was filtered andwashed consecutively with dichloromethane, IQ % methanol indichloromethane, dichloromethane and anhydrous diethyl ether. The CPGwas suction dried for 1 hour, then recovered from the funnel and placedunder hard vacuum for 2 hours. The loaded CPG was capped with 25% aceticanhydride in pyridine (100 mL) for three hours. The CPG was filtered andthe same washing procedure described earlier was repeated. The CPG wassuction dried for 1 hour and dried under high vacuum overnight to affordcompound 125 (10.2 g, 71 μmol/g).

Compound 127: 5-Hexenol (6.0 mL, 50.5 mmol) was dissolved indichloromethane (120 mL). To this solution triethylamine (14 mL, 151.5mmol) was added. The reaction mixture stirred under argon for 30 minutesat 0° C. Then DMTr-Cl (18 g, 53.0 mmol) was added to the solution at 0°C. The mixture stirred under vacuum followed by argon, and stirring wascontinued under argon at room temperature for 4 hours. The reactionmixture was washed with water twice followed by saturated sodiumchloride. The organic layer was dried over sodium sulfate, filtered andevaporated to dryness to afford compound 127 (Rf=0.85 in 25% EtOAc inHexane) (18.9 g, 95%) as a yellow oil. NMR (400 MHz, DMSO-d₆): δ7.42-7.03 (m, 7H), 7.01-6.74 (m, 6H), 5.85-5.63 (m, 1H), 5.05-4.77 (m,2H), 3.93-3.47 (m, 6H), 2.93 (dd, J=21.1, 14.7 Hz, 2H), 2.49 (dd, J=3.5,1.7 Hz, 2H), 1.66-1.47 (m, 4H).

Compound 128: Compound 127 (5.0 g, 12.5 mmol) and sodium bicarbonate(4.2 g, 25 mmol) were mixed in dichloromethane (250 mL). The reactionmixture was stirred for 5 minutes at room temperature. Thenmeta-chloroperbenzoic acid (16 g, 31.25 mmol) was added and the mixturecontinued stirring at room temperature overnight. The reaction mixturewas quenched by the addition of sodium bisulfite (500 mg) and allowed tostir at room temperature for 30 minutes. The reaction mixture was washedwith water, saturated bicarbonate solution, water, and saturated sodiumchloride. The organic layer was dried over sodium sulfate, filtered andevaporated to dryness, to afford compound 128 (Rf=0.25 5% EtOAc inhexane) (5.06 g, 97.3%) as a yellow solid, which required no furtherpurification. ¹H NMR (400 MHz, DMSO-d₆): δ 7.48-7.08 (m, 7H), 6.88 (t,J=5.9 Hz, 6H), 3.74 (d, J=19.4 Hz, 3H), 3.32 (s, 6H), 3.04-2.77 (m, 1H),2.65-2.35 (m, 2H), 1.69-1.09 (m, 6H).

Compound 129: Compound 128 (5.0 g, 9.56 mind) was dissolved in ethanol(15 mL), 30% Ammonium hydroxide in water (3 mL) was added to thereaction mixture, which was stirred at 85° C. in an oil bath in apressure vessel overnight. The reaction mixture was evaporated todryness and then co-evaporated with toluene (10 mL) twice. The compoundwas then co-evaporated with dichloromethane (50 mL) affording crudecompound 129 (R_(f)=0.1 25% EtOAc in hexane, 5.48 g) as a brown oil,which was used without purification.

Compound 130: Compound 129 (5.48 g, crude) was dissolved indichloromethane (100 mL). The reaction mixture stirred under argon for10 minutes at 10° C. To the reaction mixture triethylamine (4.0 mL, 19.1mmol) was added dropwise as the mixture continued to stir under argonfor 20 minutes at 10° C. To the reaction mixture ethyl trifluoroacetate(5.0 mL, 28.7 mmol) was added drop wise at 10° C. The reaction mixturecontinued to stir under argon at room temperature overnight. Thereaction mixture was then washed with water twice followed by saturatedsodium chloride. The organic layer was dried over sodium sulfate,filtered and evaporated to dryness affording crude compound 130 (Rf=0.4350% EtOAc/Hex, 6.12 g). Purification of Compound 130 by silica gelcolumn chromatography by first eluting with 5% ethyl acetate in hexane(1% TEA) to remove impurities followed by 10% ethyl acetate in hexane(1% TEA) to elute product from further impurities afforded 1.51 g (30%from compound 129) as a white foam. ¹H NMR (400 MHz, DMSO-d₆): δ7.69-7.12 (m, 7H, 1 NH, D₂O exchangeable), 7.10-6.70 (m, 6H), 4.12-3.48(m, 9H, 1OH, D₂O exchangeable), 3.29 (dd, J=19.7, 12.9 Hz, 2H),21.57-1.17 (m, 6H), ¹³C NMR (101 MHz, DMSO-d₆): δ 157.97 (s), 156.56(s), 156.20 (s), 145.27 (s), 136.08 (s), 129.58 (s), 127.69 (d, J=7.7Hz), 126.51 (s), 117.45 (s), 114.58 (s), 113.09 (s), 85.15 (s), 67.93(s), 62.78 (s), 54.96 (s), 45.67 (s), 40.13 (s), 39.92 (s), 39.71 (s),39.50 (s), 39.30 (s), 39.09 (s), 38.88 (s), 34.28 (s), 29.51 (s), 21.92(s).

Compound 131: Compound 130 (1.49 g, 2.8 mmol). DMAP (1.02 g, 8.4 mmol),and triethylamine (0.8 mL, 5.6 mmol) were dissolved in dichloromethane(30 mL). The reaction mixture was stirred under argon for 5 minutes.Then succinic anhydride (600 mg, 5.6 mmol) was added and the mixturecontinued stirring under argon at room temperature overnight. Thereaction mixture was diluted in dichloromethane (100 mL) then washedwith two 50 mL portions of slightly saturated sodium chloride. Theorganic layer was dried over sodium sulfate, filtered and evaporated todryness. Compound 131 (Rf=0.24 25% EtOAc in hexane) (1.77 g, 99%) wasobtained as a white foam, which required no further purification.

Compound 132: Compound 131 (1.77 g, 2.8 mmol) was dissolved inacetonitrile (120 mL). To the solution diisopropylethylamine (1.95 mL,11.2 mmol) and HBTU (2.13, 5.6 mmol) were added. The reaction mixturewas swirled until all contents were dissolved. CPG (26 g) was added tothe flask and the mixture was shaken overnight. The reaction mixturewere decanted over a sintered funnel and was washed with 1%triethylamine/dichloromethane, followed by two washes of 10% methanol indichloromethane, another wash of 1% triethylamine in dichloromethane,and anhydrous diethyl ether. The CPG was suction dried for 1 hour, thenrecovered from the funnel and placed under hard vacuum for 2 hours, thenit was capped with 25% acetic anhydride in pyridine (200 mL) and themixture was shaken for three hours. The reaction mixture was then placedover a sintered funnel and washed in the same manner as before. The CPGwas suction dried for 1 hour, removed from the funnel and placed underhard vacuum overnight. (27 g, 83 μmol/g).

Compound 134: 5-Hexenol (6.0 mL, 50.5 mmol) and sodium azide (17 g,252.5 mmol) were dissolved in DMF (120 mL). To this solutiontriethylamine (14 mL, 151.5 mmol) was added. The reaction mixturestirred under argon for 10 minutes. Then methanesulfonyl chloride (4.25mL, 50.5 mmol) was added to the solution dropwise over 20 minutes.Stirring was continued under argon at room temperature for 2 days. Thereaction mixture was decanted into iced water, which was then washedwith 5×50 mL portions of diethyl ether. The organic layer was dried oversodium sulfate, filtered and evaporated to dryness, to afford crudecompound 134 (Rf=0.67 in 5% MeOH/DCM, 6.0 g). Purification of compound134 by column chromatography by first eluting with dichloromethane toremove impurities followed by 2% methanol/dichloromethane afforded 4.6 g(72%) of a clear liquid. ¹H NMR (400 MHz, DMSO-d₆): δ 5.78 (ddt, J=16.9,10.2, 6.6 Hz, 1H), 5.16-4.74 (m, 2H), 2.68-2.14 (m, 2H), 1.64-1.27 (m,4H), 1.24-0.92 (m, 2H)

Compound 135: Compound 134 (4.5 g, 25.5 mmol) was taken in DMF (100 mL)in a pressure bottle. To this mixture NaN₃ (10 g) was added and themixture was heated at 80° C. overnight. Solids were then removed byfiltration. Volatiles were removed under vacuum and the residue wasextracted with ethyl acetate and washed with water and brine. Theorganic layer was dried over sodium sulfate and the solvent was removedto afford compound afford 134134 as pale yellow liquid (3.6 g, 82%).m/z: 132.1 (—N₂).

Compound 136: Compound 135 (2.0 g, 16 mmol) andN-methylmorpholine-N-oxide (2.25 g, 19.2 mmols) were dissolved in 10%water in acetone (60 mL). The reaction was stirred for 5 minutes at roomtemperature. Then osmium tetroxide in 10% water in acetone (1.3 mL, 0.16mmol) was added and the mixture continued stirring at room temperatureovernight. The reaction mixture was decanted over Celite and washed withtwo 50 mL portions of acetone. The reaction mixture was evaporated todryness then dissolved in 100 mL of ethyl acetate. The organic layer waswashed with a 50 mL portion of 1N HCl. The organic layer was dried oversodium sulfate, filtered and evaporated to dryness, to afford Compound136 1.70 g (67%) as a tan liquid, which required no furtherpurification.

Compound 137: Compound 136 (1.5 g, 9.40 mmols) was co-evaporated withanhydrous pyridine (10 mL) twice. Then the compound was placed underhigh vacuum overnight to dry. The compound was then taken from highvacuum and dissolved in anhydrous pyridine (60 mL). To this solution acatalytic amount of DMAP (0.11 g, 0.94 mmol) was added. The reactionmixture was stirred under argon for 30 minutes at 0° C. Then DMT-Cl(3.35 g, 10.06 mmol) was added to the solution at 0° C. The mixturestirred under vacuum followed by argon, and stirring was continued underargon at room temperature for 1.5 hours. The reaction mixture wasevaporated to dryness, and to the residue dichloromethane (100 mL) wasadded. The organic layer was washed with water twice followed bysaturated brine. The organic layer was dried over sodium sulfate,filtered and evaporated to dryness, affording crude compound 137(R_(f)=0.8 1:1 EtOAc/Hexane, 5, 3.3 g) as an orange oil, which was usedwithout purification.

Compound 138: Compound 137 (3.3 g, crude) and triphenylphosphine (1.7 g,6.48 mmol) were dissolved tetrahydrofuran. The reaction mixture wasstirred for 5 minutes at room temperature. Then water (1 mL) was addedand the mixture continued stirring at room temperature overnight. Whencompletion of the reaction was observed by TLC, the reaction mixture wasevaporated to dryness, then co-evaporated with toluene. The reactionmixture was dissolved in dichloromethane (60 mL). The reaction mixturestirred under argon for 10 minutes at 10° C. To the reaction mixturetriethylamine (1.5 mL, 10.75 mmol) was added dropwise as the mixturecontinued to stir under argon for 20 minutes at 10° C. To the reactionmixture ethyl trifluoroacetate (6.5 mL, 54.0 mmols) was added dropwiseas the mixture continued to stir under argon for 10 minutes at 10° C.The reaction mixture continued to stir under argon at room temperatureovernight. The reaction mixture was washed with water, saturatedbicarbonate solution, water, and saturated sodium chloride. The organiclayer was dried over sodium sulfate, filtered and evaporated to drynessaffording crude compound 138 (Rf=0.5 50% EtOAc/Hex, 3.40 g).Purification of the compound by column chromatography by first elutingwith 5% ethyl acetate in hexane to remove impurities followed by 15%ethyl acetate in hexane afforded 2.1 g (42% from Compound 136) as ayellow oil. ¹H NMR (400 MHz, DMSO-d₆): δ 9.36 (s, 1 NH, D₂Oexchangeable), 7.47-7.14 (m, 7H), 6.87 (d, J=8.8 Hz, 6H), 3.86-3.48 (m,7H), 3.33 (s, 1 OH, D₂O exchangeable), 3.13 (dd, J=12.1, 6.4 Hz, 4H),1.54-1.06 (m, 6H), ¹⁹F NMR (376 MHz, DMSO-d₆): δ −82.54 (s), ¹³C NMR(101 MHz, DMSO-d₆): δ 161.42 (s), 159.73 (s), 159.37 (s), 148.65 (s),139.41 (d, J=6.7 Hz), 136.65 (s), 135.74-135.30 (m), 134.92 (d, J=9.8Hz), 133.16 (s), 132.18 (d, J=11.8 Hz), 131.18 (d, J=6.0 Hz), 129.96(s), 116.51 (s), 88.50 (s), 72.39 (s), 71.10 (s), 63.20 (s), 58.43 (s),43.58 (s), 43.37 (s), 43.16 (s), 42.95 (s), 42.80-42.53 (m), 42.33 (s),36.88 (s), 31.79 (s), 25.67 (s), 17.51 (s).

Compound 139: Compound 138 (1.0 g, 1.9 mmols). DMAP (700 mg, 5.7 mmols),and triethylamine (0.55 mL, 3.8 mmols) were dissolved in dichloromethane(20 mL). The reaction mixture was stirred under argon for 5 minutes.Then succinic anhydride (380 mg, 3.8 mmol) was added and the mixturecontinued stirring under argon at room temperature overnight. Thereaction mixture was diluted in dichloromethane (50 mL) then washed withtwo 25 mL portions of slightly saturated sodium chloride. The organiclayer was dried over sodium sulfate, filtered and evaporated to dryness.Compound 139 (eluent 1:1 EtOAc/Hexane) 1.18 g (99%) was obtained as apink oil, which required no further purification.

Compound 140: Compound 139 (1.18 g, 1.9 mmols) was dissolved inacetonitrile (60 mL). To the solution diisopropylethylamine (1.3 mL, 7.6mmol) and HBTU (1.43 g, 3.8 mmol) were added. The reaction mixture wasswirled until all the contents were dissolved. CPG (12 g) was added tothe flask and the mixture was shaken overnight. The CPG compound andreaction mixture were decanted over a sintered funnel. The reactionmixture was washed with 1% triethylamine in dichloromethane, followed bytwo washes of 10% methanol/dichloromethane, another wash of 1%triethylamine in dichloromethane, and anhydrous diethyl ether. The CPGwas suction dried for 1 hour, then recovered from the funnel and placedunder high vacuum for 2 hours. The CPG was capped with 25% acetic,anhydride/pyridine (100 mL) and the mixture was shaken for three hours.The reaction mixture were placed over a sintered funnel and washed inthe same manner as before. The CPG 140 thus obtained was suction driedfor 1 hour, removed from the funnel and placed under high vacuumovernight (12 g, 77 μmol/g).

Compound 142: To a stirred solution of (R)-glycidol (2.3 g, 31 mmol) inDCM was added triethylamine (5 mL, 36.5 mmol) followed by the additionof a 1 M solution of DMTrCl (10.66 g, 31.5 mmol) in DCM at roomtemperature. The reaction was left to stir until there was no startingmaterial as shown by TLC. A few drops of MeOH was added to hydrolyze anyunreacted DMTrCl and the mixture was stirred for 10 minutes. The productwas washed with H₂O, brine, and dried over Na₂SO₄. The product waspurified by column chromatography using a gradient of Hexane/EtOAc (9:1)to afford 6 g (52%) of the pure product 142. ¹H NMR (400 MHz, DMSO-d₆):δ 7.38 (d, J=7.6 Hz, 2H), 7.31 J=7.6 Hz, 2H), 7.27-7.12 (m, 5H), 6.87(d, J=6.1 Hz, 4H), 3.72 (s, 6H), 3.24 (dd, J=10.9, 2.4 Hz, 1H),3.15-3.08 (m, 1H), 2.86 (dd, J=10.9, 6.0 Hz, 1H), 2.70 (t J=4.6 Hz, 1H),2.54 (m, 1H). ¹³C NMR (101 MHz, DMSO-d₆): δ 158.07, 144.75, 135.48,135.45, 129.60, 127.85, 127.59, 126.69, 113.46, 113.21, 85.46, 64.52,55.01, 50.37, 43.59.

Compound 144: To a stirred solution of N1,N6-dimethylhexane-1,6-diamine143 (4.225 g, 29 mmol) and K₂CO₃ (0.15 g, 1 mmol) dissolved in DMF andheated to 90° C. was added drop-wise compound 142 (5.06 g, 13.5 mmol).The reaction mixture was stirred overnight until no compound 142remained. The reaction was quenched with ice, extracted with DCM, driedover Na₂SO₄, and purified by column chromatography using a gradient ofDCM (2.5% NEt₃) MeOH (30%) to afford 7.8 g (63%) of pure compound 144.¹H NMR (500 MHz, DMSO-d₆): δ 7.40 (d, J=7.6 Hz, 2H), 7.35-7.14 (m, 7H),6.88 (d, J=8.5 Hz, 4H), 4.12 (s, 1H), 3.80-3.64 (s, 6H), 3.37 (m, 2H),3.11-2.89 (m, 5H), 2.85 (dt, J=15.1, 7.6 Hz, 1H), 2.82-2.76 (m, 2H),2.69 (s, 3H), 2.48-2.38 (s, 3H), 1.61 (m, 4H), 1.28 (m, 4H). ¹³C NMR(DMSO-d₆): δ 158.03, 144.82, 135.54, 135.48, 129.72, 127.79, 127.69,126.63, 113.15, 85.41, 65.77, 64.74, 58.49, 55.03, 54.92, 52.02, 47.83,45.17, 40.00, 39.92, 39.83, 39.76, 39.66, 39.50, 39.33, 39.16, 39.00,32.11, 25.50, 25.40, 24.99, 23.29, 8.37, 7.23.

Compound 146: (OBz) GalNAc Acid 145 (6.95 g, 13.3 mmol), HOB((3.59 g,26.6 mmol), HBTU (5.04 g, 13.3 mmol), and DIPEA (5 mL, 28.5 mmol) werestirred in anhydrous DMF (80 mL) for 15 minutes. To this solution wasadded Compound 144. The mixture was stirred for 45 minutes until nostarting material remained. The product was dissolved in ethyl acetate(100 ml) and the organic layer was washed with H₂O, saturated NaHCO₃,brine, dried over Na₂SO₄ then purified by column chromatography using agradient of EtOAc (1% NEt₃: MeOH (15%) to afford 5.35 g (36%) of pureCompound 146. 1H NMR (400 MHz, DMSO-d₆): δ 8.08-7.85 (m, 4H), 7.82-7.11(m, 20H), 6.86 (d, J=8.8 Hz, 4H), 5.76 (d, J=3.3 Hz, 1H), 5.36 (dt,J=26.7, 13.3 Hz, 1H), 4.75 (d, J=8.5 Hz, 1H), 4.61-4.40 (m, 3H), 4.32(ddd, J=25.2, 16.9, 9.1 Hz, 2H), 3.88-3.60 (m, 1H), 3.71 (s, 6H),3.63-3.45 (m, 1H), 3.18 (dd, J=14.6, 7.4 Hz, 2H), 3.03-2.81 (m, 4H),2.74 (s, 3H), 2.42-2.29 (m, 1H), 2.31-2.12 (m, 5H), 2.07 (s, 3H), 1.69(s, 3H), 1.60-0.99 (m, 12H). ¹³C NMR (DMSO-d₆): δ 171.45, 171.37,170.36, 170.29, 169.32, 165.19, 165.14, 164.86, 157.91, 145.23, 145.22,136.01, 135.98, 133.75, 133.49, 133.45, 129.70, 129.57, 129.20, 129.15,129.02, 128.99, 128.95, 128.68, 128.57, 127.78, 127.62, 126.45, 113.10,112.98, 100.85, 85.01, 71.88, 69.98, 68.80, 68.73, 67.94, 67.72, 66.39,63.45, 62.08, 60.72, 59.72, 57.68, 57.64, 54.96, 54.88, 49.72, 48.89,46.60, 42.83, 40.09, 40.00, 39.92, 39.83, 39.76, 39.67, 39.59, 39.50,39.33, 39.17, 39.00, 34.67, 32.69, 32.17, 31.51, 30.14, 28.65, 28.54,27.90, 26.76, 26.74, 26.48, 26.22, 26.06, 22.67, 21.41, 21.11, 20.73,20.69, 18.58, 14.06, 13.52.

Compound 147: To a stirred solution of Compound 146 (1.136 g, 1 mmol)and DIPEA (1 mL. 6 mmol) in DCM was added succinic anhydride (220 mg,2.2 mmol). The reaction was stirred overnight until no starting materialremained. The product was washed with H₂O, then brine, and dried overNa₂SO₄ to afford 850 mg (69%) of Compound 147. ¹H NMR (500 MHz,DMSO-d₆): δ 8.11 (dd, J=15.3, 9.3 Hz, 1H), 7.98-7.84 (m, 4H), 7.73-7.16(m, 19H), 6.87 (d, J=8.7 Hz, 4H), 5.74 (d, J=3.1 Hz, 1H), 5.36 (dd,J=11.1, 2.6 Hz, 1H), 5.01 (s, 1H), 4.76 (dd, J=8.5, 2.6 Hz, 1H), 4.45(m, 2H), 4.37-4.23 (m, 2H), 3.71 (s, 6H), 3.54 (dd, J=13.7, 10.4 Hz,1H), 3.21-3.02 (m, 4H), 3.02-2.93 (m, 4H), 2.87 (s, 2H), 2.73 (s, 1H),2.55-2.30 (m, 8H), 2.30-2.12 (m, 5H), 2.07 (s, 3H), 1.68 (s, 3H),1.58-1.07 (m, 8H). ¹³C NMR (126 MHz, DMSO-d₆): δ 173.83, 172.03, 171.45,171.38, 169.31, 165.19, 165.13, 164.85, 158.01, 144.84, 135.54, 133.74,133.45, 129.57, 129.19, 129.14, 129.02, 128.99, 128.94, 128.67, 128.56,127.75, 127.59, 126.59, 113.10, 100.84, 85.14, 71.92, 70.73, 69.97,68.72, 67.93, 63.16, 62.09, 57.39, 57.33, 56.90, 54.97, 49.69, 48.89,47.86, 46.59, 42.65, 40.00, 39.92, 39.83, 39.76, 39.67, 39.59, 39.50,39.33, 39.16, 39.00, 38.23, 34.68, 32.72, 32.68, 32.15, 31.49, 30.09,29.89, 29.84, 28.62, 28.53, 27.89, 26.72, 26.34, 26.16, 26.01, 22.66,21.40, 21.09, 20.46, 16.64

Compound 148: Compound 147 (0.85 g, 0.64 mmol). CPG (5 g, 0.67 mmol),HBTU (0.485 g, 1.3 mmol), and DIPEA (0.4 mL, 2 mmol) were dissolved inacetonitrile and shaken for 2 hours. The product was filtered, washedwith DCM, a solution of DCM:MeOH (9:1), and then dried. The solid wasthen shaken with a solution of pyridine:acetic anhydride (35%) for 3hours for capping. The product was filtered, washed with DCM, DCM:MeOH(9:1), hexanes, DCM, and then dried under vacuum to afford 5.2 g (70.4μmol/g loading) of Compound 148.

Compound 149: Compound 146 (2.3 g, 2.025 mmol) was azeotroped inpyridine 3× to ensure removal of all moisture, and from this pointforward, kept under argon. All solvents used were degassed with argon.To a stirred solution of Compound 4 and DIPEA (1.3 mL. 7.2 mmol) inpyridine at C was added 2-cyanoethyl diisopropylchlorophosphoramidite (1g, 4.2 mmol). The mixture was stirred until TLC indicated elimination ofall starting materials. The reaction mixture was concentrated, dissolvedin a solution of EtOAc:NEt₃ (1%):DCM (20%), and run through a quickfiltration column of silica gel using EtOAc:NEt₃ (3%):DCM (25%) in orderto filter off any salts. The filtrate was concentrated, dissolved againin EtOAc, and heptane was added until the product oiled out. Thesupernatant layer was decanted and the product was concentrated. Theproduct was again dissolved in EtOAc and added drop-wise to a solutionof 0° C. heptanes until it precipitated. The heptanes were decanted andthe solid was dried to afford 2.2 g (79%) of Compound 149. ³¹P NMR (162MHz, CD₃CN) δ 148.03, 147.94, 1H NMR (400 MHz, CD₃CN): δ 8.00-7.95 (m,4H), 7.80-7.76 (m, 2H), 7.69-7.42 (m, 10H), 7.38-7.18 (m, 10H),6.89-6.66 (m, 5H), 5.83 (d, J=3.0 Hz, 1H), 5.45-5.37 (m, 1H), 4.80 (d,J=8.6 Hz, 1H), 4.51 (ddd, J=10.7, 6.4, 2.3 Hz, 1H), 4.41-4.31 (m, 3H),3.77-3.75 (m, 5H), 3.31-3.07 (m, 4H), 2.93-2.34 (m, 8H), 2.32-2.18 (m,5H), 2.14 (m, 4H), 1.94 (dt, J=4.9, 2.5 Hz, 3H), 1.77-1.73 (m, 3H),1.64-1.56 (m, 4H), 1.36-1.10 (n, 16H), 1.09 (d, J=6.8 Hz, 2H), 0.99 (d.J=6.2 Hz, 1H). ¹³C NMR (101 MHz, CD₃CN): δ 173.16, 171.09, 166.76,166.71, 166.43, 159.80, 159.75, 146.11, 137.19, 136.98, 136.93, 134.74,134.45, 134.36, 131.12, 131.06, 130.87, 130.59, 130.56, 130.50, 130.46,130.38, 129.94, 129.66, 129.59, 129.16, 129.07, 128.97, 128.90, 128.84,128.78, 127.96, 127.90, 118.73, 118.37, 114.19, 114.14, 114.06, 114.01,101.98, 87.45, 87.15, 73.13, 71.75, 70.13, 69.21, 63.22, 59.00, 56.00,55.98, 51.70, 50.42, 48.02, 46.06, 43.01, 42.79, 35.70, 33.56, 32.93,32.69, 29.84, 29.75, 29.19, 28.25, 28.09, 27.96, 27.87, 27.50, 27.36,25.12, 23.48, 23.31, 23.18, 22.55, 22.28, 20.94, 20.89, 20.66, 14.47,2.21, 2.02, 1.81, 1.61, 1.40, 1.19, 0.99, 0.89, 0.78.

Example 49

3,4-Di-acetyl-6-mesyl GalNAc Ester 2: To a solution of 6-hydroxyderivative 1 (2.00 g, 4.3 mmol) in anhydrous DCM (20 mL) were addedconsecutively DIEA (1.2 mL, 6.5 mmol), and mesyl chloride (0.5 mL, 6.5mmol) under Ar atmosphere. The mixture was stirred at room temperatureovernight, then quenched by addition of 5% aq. NaCl (60 mL). The organicphase was separated, dried over anhydrous sodium sulfate and evaporatedunder reduced pressure. The residue was co-evaporated once withanhydrous ACN, and the remaining foamy amorphous solid was dried underhigh vacuum overnight to afford 2.38 g (quantitative) of crude compound2 that was used in the next step without further purification. MS (inAcOEt): (+) mode: 484 (M-t-Bu); (−) mode: 598 (M+AcOH), ¹H¹ NMR (400MHz). DMSO-d₆, J (Hz): 1.38 (s, 9H); 1.47 (m, 4H); 1.76 (s, 3H); 1.88(s, 31-1); 2.10 (s, 3H); 2.16 (t, 2H, J=7.1); 3.18 (s, 3H); 3.41 (m,1H), 3.71 (m, 1H); 3.87 (q, 1H, J=8.9); 4.10 (m, 1H); 4.19 (m, 2H); 4.50(d, 1H, J=8.5); 4.96 (dd, 1H, 3.4, J₂=11.2); 5.25 (d, 1H, J=2.8); 1.80(d, 1H, J=9.2).

Unprotected 6-mesyl GalNAc Ester 3: To a cold (0° C.) solution of3,4-diacetyl derivative 2 (1.29 g, 2.2 mmol) in anhydrous WON (10 mL)was added a 25 wt. % solution of MeONa in MeOH (0.05 mL, 0.22 mmol)under an argon atmosphere. The mixture was stirred at 0° C. for 2.5 h,quenched by addition of triethylamine hydrochloride (34 mg, 0.25 mmol),and evaporated in vacuum to afford 1.20 g (quantitative) of crudecompound 3 that was used in the next step without further purification.MS (in MeOH): (+) mode: 400 (M—t-Bu); (−) mode: 490 (M+Cl). H¹ NMR (400MHz), DMSO-d₆; J (Hz): 1.37 (s, 9H); 1.46 (m, 4H); 1.78 (S, 3H); 2.16(t, 2H, J=7.5); 3.17 (s, 3H); 3.36 (m, 1H), 3.46 (m, 1H); 3.67 (m, 4H);4.28 (m, 3H); 4.72 (d, 1H. J=6.2): 4.85 (d, 1H. J=4.0); 7.63 (d, 1H.J=9.0).

3,4-Isopropyliden-6-mesyl GalNAc Ester 4: The residue from the previousstep containing crude compound 3 (1.20 g, 2.2 mmol) was dissolved in amixture of 2,2-dimethoxypropane (10 mL) and acetone (2 mL), followed byaddition of methanesulfonic acid (2 drops). The mixture was stirred atroom temperature for 2.5 hours, neutralized by addition of triethylamine(4 drops), and partitioned between ethyl acetate and saturated sodiumbicarbonate. The organic phase was separated, washed with saturatedNaCl, dried over anhydrous sodium sulfate, evaporated in vacuum, theresidue was co-evaporated with anhydrous ACN, and dried under highvacuum to afford 1.10 g (quantitative) of crude compound 4 that was usedin the next step without further purification. MS (in AcOEt): (+) mode:496 (M), 440 (M—t-Bu); (−) mode: 530 (M+Cl), 554 (M+AcOH); H¹ NMR (400MHz), DMSO-d₆, J (Hz): 1.23 (s, 3H); 1.38 (s, 9H); 1.40 (s, 3H); 1.46(m, 4H); 1.79 (s, 3H); 2.16 (t, 2H, J=7.0); 3.21 (s, 3H); 3.67 (m, 1H),3.55 (m, 1H); 3.68 (m, 1H); 4.14 (m, 3H); 4.27 (dd, 1H, J₁=8.4,J₂=10.7); 4.36 (d, 1H, J=8.7); 4.42 (dd, 1H, J₁=3.3, J₂=10.9); 7.86 (d,1H, J=9.0).

6-(1-Imidazolyl) Ester 5a: A solution of mesylate compound 4 (200 mg,0.4 mmol), imidazole (136 mg, 2 mmol), and DBU (0.075 mL, 0.5 mmol) inanhydrous DMA (3 mL) was heated at 140° C., under and argon atmospherefor 23 hours. The mixture was cooled to room temperature, diluted with a1:1 mixture of saturated ammonium chloride and water (40 mL), andextracted with AcOEt. The organic phase was separated, washed twice withsaturated brine, dried over anhydrous sodium sulfate, evaporated and theresidue was chromatographed over a column of silica gel with gradient ofMeOH in AcOEt (0-50%) to afford 74 mg (40%) of compound 5a. MS (inAcOEt): (+) mode: 468 (M); (−) mode: 502 (M+Cl), 526 (M+AcOH). ¹H NMR(400 MHz), ACN-d₃, J (Hz): 1.29 (s, 3H); 1.41 (s, 9H); 1.48 (s, 3H);1.50 (m, 4H); 1.85 (s, 3H); 2.16 (t, 2H, J=7.0); 3.31 (dt, 1H, J₁=6.2,J₂=10.0); 3.64 (m, 2H); 3.99 (m, 1H); 4.03 (dd, 1H, J₁=2.0, J₂=5.1);4.20 (m, 3H); 4.30 (d, 1H, J=8.8); 6.67 (d, 1H, J=9.2); 6.92 (s split,1H); 7.08 (s split, 1H); 752 (s split, 1H).

6-(3-[3-Methoxyphenyl]-imidazolyl-1) Ester 5b: A solution of mesylatecompound 4 (300 mg, 0.6 mmol), 2-(3-methoxyphenyl)imidazole (520 mg, 3mmol), and DBU (0.14 mL, 0.9 mmol) in anhydrous DMA (4 mL) was heated at140° C., under an argon atmosphere for 76 hours. The mixture was cooledto room temperature, diluted with a 1:1 mixture of saturated ammoniumchloride and water (40 mL), and extracted with AcOEt. The organic phasewas separated, washed consecutively with 5% aq. NaCl, saturated NaCl,dried over anhydrous sodium sulfate, evaporated. The residue waschromatographed over a column of silica gel with gradient of MeOH inAcOEt (0-30%) to afford 63 mg (18%) of compound 5b. MS (in AcOEt): (+)mode: 574 (M); (−) mode: 608 (M+Cl), 532 (M+AcOH). ¹H NMR (400 MHz),ACN-d₃, J (Hz): 1.31 (s, 3H); 1.40 (s, 9H); 1.47 (m, 4H); 1.50 (s, 3H);1.85 (s, 3H); 2.13 (t, 2H, J=7.1); 3.33 (m, 1H); 3.65 (m, 2H); 3.80 (s,3H); 4.03 (m, 1H); 4.08 (dd, 1H, J₁=2.1, J₂=5.2); 4.20 (m, 3H); 4.32 (d,1H, J=8.8); 6.48 (d, 1H, J=9.2); 6.77 (ddd, 1H, J₁=1.2, J₂=2.5, J₃=8.1);7.52 (t, 1H, J=8.2); 7.33 (m, 2H); 7.48 (d, 1H, J=1.3); 7.56 (d, 1H,J=1.3).

Deprotected Imidazolyl Derivatives 6a and 6b: Protected derivative 5a or5b (0.11 mmol) was dissolved in 98% formic acid (2 mL), and water (0.05mL) was added. The solution was allowed to stay at room temperatureovernight, the formic acid was evaporated in vacuum, and the residue wasco-evaporated twice with 1:1 mixture of ethanol and toluene (6 mL). Theresidue was dissolved in methanol (3 mL), triethylamine (0.15 mL) wasadded and the solution was stirred at 65° C. for 4 hours. Methanol wasremoved in vacuum, and the residue was co-evaporated 3 times with 3 nitof pyridine to afford 49 and 73 mg of crude compounds 6a and 6brespectively. The products were further purified by crystallization fromacetonitrile—methanol mixtures. 6a: MS (in MeOH): (+) mode: 472 (MH+):(−) mode: 370 (M—H⁺). ¹H NMR (400 MHz), DMSO-d₆, J (Hz): 1.44 (m, 4H);1.78 (s, 3H); 2.17 (t, 2H, J=7.0); 3.26 (m 1H); 3.43 (dd, 1H, J₁=3.0,J₂=10.6); 3.51 (d, 1H, J=2.8); 3.57 (m, 2H); 3.70 (q, 1H, J=10.4); 4.11(m, 2H); 4.18 (d, 1H, J=8.4); 6.86 (s, 1H); 7.14 (s, 1H); 7.59 (m, 2H);12.0 (s broad, COOH). 6b: MS (in MeOH): (+) mode: 478 (MH⁺); (−) mode:476 (M—H⁺). H¹ NMR (400 MHz), DMSO-d₆. J (Hz): 1.44 (m, 4H); 1.78 (s,3H); 2.16 (t, 2H, J=7.0); 3.28 (m, 1H); 3.45 (m, 1H); 3.55 (s broad,1H); 3.62 (m, 2H); 3.71 (m, 1H); 3.76 (s, 3H); 4.15 (m, 2H); 4.22 (d,1H, J=8.4); 4.71 (s broad, 1H, OH); 4.92 (s broad, 1H, OH); 6.73 (ddd,1H, J₁=1.4, J₂=2.5, J₃=8.0); 7.23 (t, 1H, J=8.0); 7.28 (m, 2H); 7.63 (m,3H), 12.0 (s broad, COOH).

Example 50

Histamine Derivative 8: A suspension of NHS ester compound 7 (200 mg,0.33 mmol) and histamine base (52 mg, 0.47 mmol) in anhydrous DCM (3 mL)and pyridine (0.05 mL) was stirred at room temperature for 5 days. Thereaction was quenched by addition of saturated aqueous sodiumbicarbonate and the product was extracted with ethyl acetate. Theorganic phase was separated, washed with saturated brine, and dried overanhydrous sodium sulfate. The product compound 8 was isolated by columnchromatography on silica gel using a gradient of MeOH in DCM (0-30%).Yield: 64 mg, 32%. MS (in MeOH): (+) mode: 599 (M); (−) mode: 633(M+Cl). ¹H NMR (400 MHz), ACN-d₃. J (Hz); 1.41 (s, 9H); 1.54 (m, 4H);1.83 (s, 3H); 1.91 (s, 3H); 2.09 (s, 3H); 2.18 (t, 2H, J=7.0); 2.69 (t,2H, J=6.5); 3.29 (m, 2H); 3.47 (m, 1H); 3.78 (m, 1H); 3.87 (t, 1H,J=6.3); 3.94 (q, 1H, J=11.0); 4.04 (m, 2H); 4.51 (d, 1H, J=8.5); 5.00(dd, 1H, J₁=3.2, J₂=11.2); 5.26 (d, 1H, J=2.9); 5.91 (s broad, 1H); 6.40(d, 1H, J=9.4); 6.81 (s broad, 1H); 7.49 (s broad, 1H).

Deprotected Histamine Derivative 9: A solution of protected histaminederivative 8 (63 mg, 0.11 mmol) in 98% formic acid (2 mL) was maintainedat room temperature overnight, then diluted with toluene (10 mL) andevaporated in vacuum. The residue was co-evaporated once with a MeOH-ACNmixture and once with a MeOH-pyridine mixture. The product was dried inhigh vacuum, dissolved in methanol (3 mL) and triethylamine (0.3 mL) wasadded. The mixture was stirred at 65° C. overnight, filtered,evaporated, and the residue was co-evaporated 3 times with pyridine (3mL) one time with ACN (5 mL), and dried under high vacuum to afford 50mg (quant) of compound 9 as a highly hygroscopic white solid. MS (inMeOH): (−) mode: 457 (M−H). ¹H NMR (400 MHz). DMSO-d₆, J (Hz): 1.46 (m,4H); 1.78 (s, 3H); 2.18 (t, 2H, J=7.3): 2.61 (t, 2H, J=7.6); 3.18 (q,2H, J=6.9); 3.34 (m, 2H); 3.44 (dd, 1H, J₁=3.0, J₂=10.6); 3.51 (t, 1H,J=5.8): 3.60 (d, 1H, J=2.4); 3.68 (m, 2H); 4.04 (d, 2H, J=5.8); 4.23 (d,1H, J=8.4); 4.67 (s broad, 2H); 6.77 (s, 1H); 7.23 (t, 1H, J=5.5); 7.52(s, 1H); 7.60 (d, 1H, J=9.0); 12.01 (s broad, 2H).

Example 51

Synthesis of compound 102: Compound 101 (5 g, 22.6 mmol) was heated at100° C. with 5-hexen-1-ol (80 mL) and boron trifluoride diethyl etherate(0.5 mL) for 16 hours. Trituration with Et₂O afforded compound 102 (4.0g, 13.2 mmol, 58%). Molecular weight for C₁₄H₂₆NO₆ (M+H)⁺ Calc.304.1760. Found 304.2.

Synthesis of compound 103: Compound 102 (1.48 g, 4.88 mmol) was treatedin pyridine (30 mL) and Ac₂O (10 mL). Aqueous work-up and silica gelcolumn purification afforded compound 103 (1.48 g, 3.45 mmol, 70%).Molecular weight for C₂₀H₃₂NO₉ (M+H)⁺ Calc 430.2077. Found 430.2.

Synthesis of compound 104: Compound 103 (1.37 g, 3.19 mmol) was treatedwith lipase from Candida rugosa (3.43 g) in dioxane (13.7 mL) andpotassium hydrogen phthalate buffer (54.8 mL, pH=4) for 64 hours.Aqueous work-up and silica gel column purification afforded compound 104(680 mg, 1.76 mmol, 55%). ¹H NMR (400 MHz. CDCl₃) δ 5.85-5.75 (m, 1H),5.59 (d, J=9.7 Hz, 1H), 5.33-5.32 (m, 1H), 5.21 (dd, J=11.3 Hz, 3.2 Hz,1H), 5.06-4.96 (m, 2H), 4.86 (d, J=3.7 Hz, 1H), 4.63-4.57 (m, 1H), 4.01(td, J=6.5 Hz, 1.2 Hz, 1H), 3.73-0.3.62 (m, 2H), 3.51-3.39 (m, 2H), 2.19(s, 3H), 2.12-2.06 (m, 2H), 2.02 (s, 3H), 1.96 (s, 3H), 1.66-1.59 (m,2H), 1.49-1.41 (m, 2H).

Synthesis of compound 105: Compound 104 (747 mg, 1.74 mmol) was treatedwith triphenylphosphine (913 mg, 3.48 mmol), diisopropylazodicarboxylate (0.674 mL, 3.48 mL) and diphenyl phosphoryl azide(0.752 mL, 3.48 mL) in THF (17 mL) for 18 hours. Aqueous work-up andsilica gel column purification gave compound 105 (752 mg, quantitative).¹H NMR (400 MHz, CDCl₃) δ 5.85-5.75 (m, 1H), 5.56 (d, J=9.7 Hz, 1H),5.30 (d, J=2.7 Hz, 1H), 5.15 (dd, J=11.3, 3.3 Hz, 1H), 5.05-4.97 (m,2H), 4.88 (cl, J=3.7 Hz, 1H), 4.60-4.54 (m, 1H), 4.06 (dd, J=8.8, 3.6Hz, 1H), 3.77-3.71 (m, 1H), 3.49-3.40 (m, 2H), 3.13 (dd, J=12.8, 4.1 Hz,1H), 2.17 (s, 3H), 2.12-2.07 (m, 2H), 1.99 (s, 3H), 1.96 (s, 3H),1.65-1.60 (m, 2H), 1.50-1.44 (m, 2H).

Example 52

Synthesis of compound 106: Compound 105 (730 mg, 1.77 mmol) was treatedwith ruthenium (III) chloride hydrate (18 mg, 0.089 mmol) and sodiumperiodate (1.89 g, 8.85 mL) in CH₂Cl₂ (5 mL) and CH₃CN (5 mL) and H₂O (7mL) for 18 hours. Aqueous work-up and silica gel column purificationafforded compound 106 (677 mg, 1.57 mmol, 89%). Molecular weight forC₁₇H₂₇N₄O₉(M+H)⁺ Calc 431.1778. Found 431.1.

Synthesis of compound 107: Compound 106 (200 mg, 0.465 mmol) was treatedwith N-hydroxysuccinimide (80 mg, 0.698 mmol),N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (134 mg,0.698 mmol) and DIEA (0.244 mL, 1.40 mmol) in CH₂Cl₂ (3 mL) for 18hours. Aqueous work-up and silica gel column purification affordedcompound 107 (183 mg, 0.347 mmol, 75%). Molecular weight for C₂₁H₃₀N₅O₁₁(M+H)⁺ Calc 528.1942. Found 528.1.

Synthesis of compound 108: To a solution of compound 106 (227 mg, 0.527mmol) and 3-ethynylanisole (0.080 mL, 0.632 mmol) in MeOH (2 mL) wereadded a solution of THPTA (11 mg, 0.0264 mmol) and CuSO₄.5H₂O (1.3 mg,0.00527 mmol) in H₂O (0.1 mL) and a solution of sodium ascorbate (10 mg,0.0527 mmol) in H₂O (0.1 mL). The reaction mixture was stirred at roomtemperature for 18 hours. Aqueous work-up and silica gel columnpurification afforded compound 108 (264 mg, 0.469 mmol, 89%). Molecularweight for C₂₆H₃₅N₄O₁₀ (M+H)⁺ Calc 563.2353. Found 563.2.

Synthesis of compound 109: Compound 108 (233 mg, 0,414 mmol) was treatedwith N-hydroxysuccinimide (72 mg, 0.621 mmol),N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (119 mg,0.621 mmol) and DIEA (0.216 mL, 1.24 mmol) in CH₂Cl₂ (4 mL) for 18hours. Aqueous work-up and silica gel column purification affordedcompound 109 (100 mg, 0.152 mmol, 37%). Molecular weight for C₃₀H₃₈N₅O₁₂(M+H)⁺ Calc 660.2517. Found 660.2.

Synthesis of compound 110: To a solution of compound 106 (223 mg, 0.518mmol) and 2-methyl-8-(prop-2-yn-1-yloxy)quinoline (123 mg, 0.622 mmol)in MeOH (2 mL) were added a solution of THPTA (11.2 mg, 0.0259 mmol) andCuSO₄.5H₂O (1.3 mg, 0.00518 mmol) in H₂O (0.1 mL) and a solution ofsodium ascorbate (10.3 mg, 0.0518 mmol) in H₂O (0.1 mL). The reactionmixture was stirred at room temperature for 18 hours. Aqueous work-upand silica gel column purification afforded compound 110 (320 mg, 0.510mmol, 98%). Molecular weight for C₃₀H₃₈N₅O₁₀ (M+H)⁺ Calc 628.2619. Found628.2.

Synthesis of compound 111: Compound 110 (307 mg, 0,489 mmol) was treatedwith N-hydroxysuccinimide (85 mg, 0.734 mmol),N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (141 mg,0.734 mmol) and DIEA (0.256 mL, 1.47 mmol) in CH₂Cl₂ (4 mL) for 18hours. Aqueous work-up and silica gel column purification affordedcompound 111 (155 mg, 0.207 mmol, 42%). Molecular weight for C₃₄H₄₁N₆O₁₂(M+H)⁺ Calc 725.2782. Found 725.1.

Synthesis of compound 112: Compound 106 (240 mg, 0.558 mmol) was treatedin MeOH (9 mL) and Et₃N (1 mL) for 5 days to afford compound 112 (250mg, 0.558 mmol, quantitative). Molecular weight for C₁₃H₂₃N₄O₇ (M+H)⁺Calc 347.1567. Found 347.1.

Example 53

Synthesis of compound 121: Using compound 120 in place of compound 106,a procedure analogous to that described for compound 108 was followed toafford compound 121 (300 mg, 0.495 mmol, 80%). Molecular weight forC₂₉H₄₀FN₄O₉ (M+H)⁺ Calc 607.2779. Found 607.1.

Synthesis of compound 122: Using compound 120 in place of compound 106,a procedure analogous to that described for compound 108 was followed toafford compound 122 (417 mg, 0.635 mmol, 90%). Molecular weight forC₃₀H₄₀F₃N₄O₉ (M+H)⁺ Calc 657.2747. Found 657.2.

Synthesis of compound 123: Using compound 120 in place of compound 106,a procedure analogous to that described for compound 108 was followed toafford compound 123 (343 mg, 0.559 mmol, 80%). Molecular weight forC₃₀H₄₀N₅O₉ (M+H)⁺ Calc 614.2826. Found 614.2.

Synthesis of compound 124: Using compound 120 in place of compound 106,a procedure analogous to that described for compound 108 was followed toafford compound 124 (340 mg, 0.537 mmol, 79%). Molecular weight forC₂₉H₄₁N₅O₁₁ (M+H)⁺ Calc 634.2724. Found 634.0.

Synthesis of compound 125: Using compound 120 in place of compound 106,a procedure analogous to that described for compound 108 was followed toafford compound 125 (323 mg, 0.54 mmol, 83%). Molecular weight forC₂₉H₄₁N₄O₁₀ (M+H)⁺ Calc 605.2823. Found 605.2.

Synthesis of compound 126: Compound 121 (290 mg, 0.478 mmol) was treatedwith formic acid (10 mL) for 18 hours. After removing the solvent,silica gel column purification afforded compound 126 (241 mg, 0.438mmol, 92%). Molecular weight for C₂₅H₃₂FN₄O₉ (M+H)⁺ Calc 551.2153. Found551.0.

Synthesis of compound 127: Compound 122 (407 mg, 0.620 mmol) was treatedwith formic acid (10 mL) for 18 hours. After removing the solvent,silica gel column purification afforded compound 127 (318 mg, 0.530mmol, 85%). Molecular weight for C₂₆H₃₂F₃N₄O₉ (M+H)⁺ Calc 601.2121.Found 601.0.

Synthesis of compound 128: Compound 123 (333 mg, 0.543 mmol) was treatedwith formic acid (10 mL) for 18 hours. After removing the solvent,silica gel column purification afforded compound 128 (281 mg, 0.504mmol, 93%). Molecular weight for C₂₆H₃₂N₅O₉ (M+H)⁺ Calc 558.2200. Found558.2.

Synthesis of compound 129: Compound 124 (330 mg, 0.521 mmol) was treatedwith formic acid (10 mL) for 18 hours. After removing the solvent,silica gel column purification afforded compound 129 (277 mg, 0.480mmol, 92%). Molecular weight for C₂₅H₃₂N₅O₁₁ (M+H)⁺ Calc 578.2098. Found578.0.

Synthesis of compound 130: Compound 125 (313 mg, 0.518 mmol) was treatedwith formic acid (10 mL) for 18 h. After removing the solvent, silicagel column purification afforded compound 130 (253 mg, 0.461 mmol, 89%).Molecular weight for C₂₅H₃₃N₄O₁₀ (M+H)⁺ Calc 549.2197. Found 549.0.

Example 54

The NHS ester compounds 131, 132, 133, 134 and 135 are prepared by astandard esterification process with N-hydroxysuccinimide usingcompounds 126, 127, 128, 129 and 130, respectively. Fully deprotectedGalNAc derivatives 136, 137, 138, 139 and 140 are prepared fromcompounds 126, 127, 128, 129 and 130 by treatment with Et₃N/MeOH.

Example 55

Synthesis of 201: Compound 200 (20 g, 44.74 mmol) was stirred indichloromethane (150 EDAC (12.8 g, 65 mmol). DMAP (2 g, catalytic), andt-butanol (20 mL) were then added. The mixture was stirred for two daysat room temperature. The solvents were then removed in vacuo. Theresidue was extracted with dichloromethane (3×100 mL) and dried oversodium sulfate. The crude product was purified by silica gelchromatography using ethyl acetate and hexane to afford compound 201 (15g, 68%). Molecular weight for C₂₃H₃₇NO₁₁ Calc. 503.24. Found 526.23(M+Na).

Synthesis of Compound 202: Using a procedure similar to that describedfor the synthesis of compound 104, 10 g of compound 201 was converted tocompound 202 (7.6 g, 78%). Molecular weight for C₂₁H₃₅NO₁₀ Calc. 461.23.Found 484.25 (M+Na).

Synthesis of Compound 203: Using a procedure similar to that describedfor the synthesis of compound 105, compound 203 was synthesized (5.2 g,64%). Molecular weight for C₂₁H₃₄N₄O₉ Calc. 486.23. Found 509.24 (M+Na).

Synthesis of Compound 204: Compound 203 (6.34 g, 13.03 mmol) was stirredin formic acid (20 ml) overnight. Solvent was removed and the residuedissolved in dichloromethane and washed with water and brine. The crudeproduct was purified by silica gel chromatography using ethylacetate/hexane to afford compound 204. (4.6 g, 82%). Molecular weightfor C₁₇H₂₆N₄O₉ Calc. 430.17. Found 453.18 (M+Na).

Synthesis of Compound 205: Compound 204 (0.50 g, 1.16 mmol) wasdissolved in dichloromethane (50 mL). N-hydroxy succinimide (0.200 g,1.5 eq), EDAC and DIEA were then added and the resulting mixture wasstirred overnight. The mixture was then washed with water and brine. Thesolvent was removed and the residue was purified by filtrationchromatography using ethyl acetate/hexane to afford compound 205.

Synthesis of Compound 206: Compound 204 (1.00 g, 2.32 mmol) was stirredin a mixture of methanol/water (2:1), 1-ethynyl-3-methoxybenzene (204A)(0.367 g, 2.7 mmol), CuSO₄.xH₂O (0.050 g, catalytic amount) and sodiumascorbate (0.25 g, 1 mmol)) were then added and the mixture was stirredovernight. The solvent was removed and the residue was dissolved indichloromethane, then washed with water, brine and dried over sodiumsulfate. The crude product was purified by silica gel chromatography toafford Compound 206. Molecular weight for C₂₆H₃₄N₄O₁₀ Calc. 562.23.Found 585.22 (M+Na).

Synthesis of Compound 207: Compound 207 was prepared using a proceduresimilar to that described for the synthesis of compound 205 (320 mg,95%).

Example 56

Synthesis of Compound 207: Compound 202 (2.8 g, 6.07 mmol) was dissolvedin dichloromethane (100 mL) and the mixture was cooled in an ice-waterbath. To this mixture DSC (1.3 g, 1.5 eq) and TEA (0.7 mL) were addedand the solution was stirred overnight. The reaction mixture was thendiluted with dichloromethane and transferred to a separatory funnel. Themixture was washed with water and brine, then dried over sodium sulfate.Solvents were removed in vacuo and the residue was dried under vacuumovernight. The resulting product was used for the next reaction stagewithout any further purification.

Synthesis of Compound 208: The DSC derivative compound 207 (0.5 g, 0.83mmol) was stirred in dichloromethane (20 mL). Benzyl amine (0.100 g, 1mmol) and pyridine (5 mL) were added and the mixture was stirredovernight. Solvents were then removed in metro. The residue wasdissolved in dichloromethane and washed with water and brine. The crudeproduct was purified by silica gel chromatography usingdichloromethane/methanol to afford compound 208 (0.300 g, 65%).Molecular weight for C₂₉H₄₂N₂O₁₁ Calc. 594.28. Found 595.29 (M+).

Synthesis of Compound 209: The DSC derivative compound 207 (0.5 g, 0.83mmol) was stirred in dichloromethane (20 mL). Ethanol amine (0.07 g, 1mmol) and pyridine (5 mL) were then added and the mixture was stirredovernight. Solvents were then removed in vacuo. The residue wasdissolved in dichloromethane and washed with water and brine. The crudeproduct was purified by silica gel chromatography usingdichloromethane/methanol to afford compound 209 (0.25 g, 42%). Molecularweight for C₂₄H₄₀N₂O₁₂ Calc. 548.26. Found 549.27 (M+H).

Synthesis of Compound 210: Compound 208 (250 mg, 0.42 mmol) wasdissolved in formic acid (20 mL) and the solution was stirred overnight.Solvents were then removed under reduced pressure. The crude compoundwas dissolved in dichloromethane and washed with water and brine.Solvents were removed in vacuo and the residue was purified by silicagel chromatography using dichloromethane/methanol to afford compound 210(200 mg, 87%). This compound was then dissolved in methanol and TEA (2mL) was added. After stirring overnight at room temperature, solventswere removed in vacuo and the residue was co-evaporated with pyridinetwo times. The product was further dissolved in water then lyophilizedto afford compound 210 as white powder. Molecular weight for C₂₁H₃₀N₂O₉Calc. 454.20. Found 477.21 (M+Na).

Synthesis of Compound 211: Compound 211 was prepared from compound 209using a method similar to that used for the preparation of compound 210.Molecular weight for C₁₆H₂₈N₂O₁₀ Calc. 408.17. Found 431.20 (M+Na).

Example 57

Synthesis of Compound 213: Glutamate derivative A. (0.174 g, 1 mmol) wasdissolved in DMF (10 mL). HBTU (0.390 g, 1.05 mmol) and DIEA were addedand the mixture was stirred for few minutes at room temperature. Aminoderivative compound 212 (0.400 g, 1 mmol) in DMF was added to thissolution and stirring was continued overnight. Solvents were thenremoved under reduced pressure. The crude compound was purified bysilica gel chromatography using dichloromethane/methanol to affordcompound 213 (0.350 g, 57%). Molecular weight for C₂₃H₄₀N₄O₁₀ Calc.532.27. Found 555.28 (M+Na).

Synthesis of Compound 214: Compound 213 (300 mg, 0.56 mmol) wasdissolved in formic acid and the mixture was stirred overnight at roomtemperature. The solvent was removed in vacuo and the residueco-evaporated with toluene two times. This residue was dissolved inwater and lyophilized to afford compound 214 (200 mg, 74%) as a whitepowder. Molecular weight for C₁₉H₃₂N₄O₁₀ Calc. 476.21. Found 477.20(M+H).

Synthesis of Compound 215: Compound 215 was prepared using a similarprocedure from compound B (140 mg). Molecular weight for C₂₇H₄₈N₄O₁₀Calc. 588.34. Found 611.33 (M+Na).

Synthesis of Compound 216: Compound 216 was prepared from compound 215using a similar procedure used for preparing compound 214 (0.125 g, 35%)Molecular weight for C₂₃H₄₀N₄O₁₀ Calc. 532.27. Found 555.25 (M+Na).

Synthesis of Compound 217: Compound 216 was prepared from compound C andamino derivative 212 (0.250 mg, 65%) Molecular weight for C₂₅H₄₁N₅O₉Calc. 555.29. Found 556.31 (M+H).

Synthesis of Compound 218: Compound 218 was prepared from compound 217using a similar procedure used for preparing compound 214 (0.140 mg,45%) Molecular weight for C₂₁H₃₃N₅O₉ Calc. 499.23. Found 500.25 (M+H).

Synthesis of Compound 219: Compound 219 was prepared from compound D andamino derivative 212 using a similar procedure used for preparingcompound 213 (0.525 g, 43%). Molecular weight for C₂₄H₄₃N₃O₉ Calc.517.30. Found 518.28 (M+H).

Synthesis of Compound 220: Compound 220 was prepared from compound 219using a similar procedure used for preparing compound 214 (95 mg, 26%).Molecular weight for C₂₀H₃₅N₃O₉ Calc. 461.24. Found 462.26 (M+H).

Synthesis of Compound 221: Compound 221 was prepared from compound fromcompound E and amino derivative compound 212. (0.320 g, 65%) Molecularweight for C₂₁H₃₇N₃O₉ Calc. 475.25. Found 476.23 (M+H).

Synthesis or Compound 222: Compound 222 was prepared from compound 221using a similar procedure used for preparing compound 214 (85 mg, 56%)Molecular weight for C₁₇H₂₉N₃O₉ Calc. 419.19. Found 420.20 (M+Na).

Example 58

Compound 58 is prepared in a manner similar to that reported inInternational Publication No. WO 96/39411. O-glycosylation followed byhydrolysis affords compounds 60 and 63. The NHS ester compounds 61 and64 are prepared by a standard esterification with NHS. The acetyl groupsof compound 65 are removed selectively and the resulting hydroxyl groupsare protected by benzyl groups to afford compound 66. Oxidative cleavageof the terminal alkene affords compound 67. Esterification followed byhydrogenation affords compounds 61 and 64. O-glycosylation of compound58 followed by oxidation and hydrolysis affords compound 60.Esterification of compound 60 affords compound 61.

Trifluoromethyl acetamide (TFA) protected galactosamine (GalN-TFA) NHSesters are coupled with amine-containing oligonucleotides (compounds 69and 71) to generate Gal-TFA containing oligonucleotides (compounds 70and 72) in a post-synthetic approach.

Example 59

Synthesis of Compound 5009: To a stirred solution of compound 5008 (25.0g, 83 mmol) in pyridine (100 mL) was added TsCl (19.8 g, 103.7 mmol).The resulting mixture was stirred overnight (14 hours) at roomtemperature. The solvent was concentrated and the product was extractedwith ethyl acetate (3×50 mL), washed with water, brine and dried overanhydrous Na₂SO₄. Concentration of the solvent afforded crude compound5009 (25 g). LCMS Calculated for C₂₁H₂₉NO₈S: 455.16 (M⁺). Found: 456.1(M⁺+1), 478.0 (M⁺+Na⁺).

Synthesis of Compound 5010: To a stirred solution of compound 5009 (19.0g, 41.7 mmol) in DMF (200 mL) was added NaN₃ (16 g, 246 mmol). Theresulting mixture was stirred for 3 days at 80° C. Another 8 g of NaN₃was then added and the solution was heated to 100° C. for 14 hours. CThe solvent was concentrated and the product was extracted with ethylacetate (3×50 mL), washed with water, brine and dried over anhydrousNa₂SO₄. Concentration of the solvent afforded crude compound 5010 whichwas purified by column chromatography (5 g, 37%). LCMS Calculated forC₁₄H₂₂N₄O₅: 326.35 (W). Found: 327.1 (M⁺+1).

Synthesis of Compound 5011: To a stirred solution of LAH (139 mg, 3.52mmol) in THF (10 mL) was added drop wise a solution of compound 5010(574 mg, 1.76 mmol) in THF (10 mL) at 0° C. The mixture was stirredovernight (14 hours) at room temperature. The reaction mixture wasquenched with 1 mL of water followed by filtration over Celite andwashing with ethyl acetate (25 mL). Concentration of the solventafforded the crude product (0.5 g) which was dissolved in DCM (0 mL) andadded to a stirred solution of glutaric anhydride (251 mg, 2.2 mmol).The reaction mixture was stirred for 14 hours at room temperature.Concentration of the solvent afforded the crude acid (285 mg) which wasdissolved in 20 mL of 2N HCl in diethyl ether and stirred 3 hours.Concentration of the solvent afforded compound 5011 (200 mg). LCMSCalculated for C₁₆H₂₆N₂O₈: 374.39 (M⁺). Found: 377.1 (M⁻−1).

Synthesis of Compound 5012: To a stirred solution of compound 5011 (200nig, 0.53 mmol) and NHS (112 mg, 1.06 mmol) in DMF (10 mL) was added DCC(218 mg, 1.06 mmol). The mixture was stirred 14 hours at roomtemperature. 20 mL of ethyl acetate was then added Filtration of thesolid afforded compound 5012 (150 mg, 60%). LCMS Calculated forC₂₀H₂₉N₃O₁₀: 471.46 (M+). Found: 506 (M⁻+Cl⁻).

Example 60: Trivalent GalNAc-Conjugated Pseudouridine Building Blocks

Synthesis of Compound 143: To a solution of trivalent GalNAc acidcompound 142 (1.80 g, 0.898 mmol) in DMF (12 mL), HBTU (341 mg, 0.898mmol) and i-Pr₂NEt (0.568 mL, 3.26 mmol) were added. After 10 minutes,compound 141 (500 mg, 0.816 mmol) was added to the solution and themixture was stirred overnight. After removing the DMF in vacuo, theresidue was extracted with CH₂Cl₂ and saturated aqueous NaHCO₃. Theorganic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated. The crude product was purified by silica gel columnchromatography (0-15% MeOH in CH₂Cl₂) to afford compound 143 (1.75 g,0.673 mmol, 82%). ¹H NMR (400 MHz, DMSO-d₆) δ 11.35 (s, 1H), 7.96 (s,1H), 7.84-7.81 (m, 6H), 7.76-7.72 (m, 4H), 7.50 (d, =0.9 Hz, 1H), 6.98(s, 1H), 5.21 (d, J=3.4 Hz, 3H), 4.96 (dd, 3=11.2, 3.4 Hz, 3H), 4.53 (s,1H), 4.48 (d, J=8.5 Hz, 3H), 4.35-4.33 (m, 1H), 4.17 (d, J=4.5 Hz, 1H),4.05-3.83 (m, 17H), 3.73-3.64 (m, 3H), 3.57-3.52 (m, 12H), 3.43-3.38 (m,3H), 3.06-3.00 (m, 16H), 2.44-2.39 (m, 2H), 2.27 (1, J=6.4 Hz, 6H), 2.10(s, 9H), 2.04 (t, J=7.3 Hz, 9H), 1.99 (s, 9H), 1.89 (s, 9H), 1.77 (s,9H), 1.52-1.42 (m, 22H), 1.21 (s, 13H), 1.01 (s, 9H), 0.98 (s, 9H), 0.89(s, 9H), 0.12 (s, 3H), 0.079 (s, 3H).

Synthesis of Compound 144: Hydrogen fluoride-pyridine (˜70% HF, 0.173mL, 6.66 mmol) was diluted in pyridine (2 mL) with cooling at 0° C. Theresulting solution was added to a solution of compound 143 in CH₂Cl₂ (20mL) at 0° C. and the mixture was stirred at 0° C. for 2 hours. Thereaction solution was diluted in CH₂Cl₂ and washed with saturatedaqueous NaHCO₃ then dried over anhydrous Na₂SO₄. After evaporation ofthe volatiles, the crude product was dried in vacuo to afford its diolas a white foam. To a solution of this material in pyridine (15 mL),DMTrCl (691 mg, 2.04 mmol) was added. The reaction mixture was stirredat room temperature for 14 hours and then evaporated. The residue wasextracted with CH₂Cl₂ and saturated aqueous NaHCO₃ then dried overanhydrous Na₂SO₄. The crude product was purified by silica gel columnchromatography (0-10% WON in C₁-12Cl₂) to afford compound 144 (3.31 g,1.20 mmol, 65%).

Synthesis of Compound 145: To a solution of compound 144 (3.25 g, 1.18mmol) in CH₂Cl₂ (20 mL) were added DMAP (432 mg, 3.54 mmol) and succinicanhydride (236 mg, 2.36 mmol). The reaction mixture was stirredovernight at room temperature. After concentration, the crude waspurified by silica gel column chromatography (8% MeOH/8% Et₃N in CH₂Cl₂)to afford compound 145 (3.03 g, 1.78 mmol, 87%). ¹H NMR (400 MHz,DMSO-d₆) δ 12.22 (brs, 1 H), 11.41 (s, 1H), 7.94 (brs, 1H), 7.85-7.81(m, 6H), 7.75-7.7 (m, 4H), 7.66 (s, 1H), 7.41-7.38 (m, 2H), 7.32-7.19(m, 9H), 6.98 (s, 1H), 6.89-6.87 (m, 4H), 5.21 (d, J=3.4 Hz, 3H), 5.07(t, J=5.1 Hz, 1H), 4.96 (dd, J=11.2, 3.4 Hz, 3H), 4.55 J=5.2 Hz, 1H),4.56-4.44 (m, 4H), 4.05-3.97 (m, 10H), 3.91-3.83 (m, 3H), 3.73 (s, 6H),3.71-3.64 (m, 4H), 3.56-3.52 (m, 14H), 3.43-3.38 (m, 3H), 3.23-3.14 (m,2H), 3.06-3.00 (m, 16H), 2.46-2.38 (m, 4H), 2.27 (t, J=6.4 Hz, 6H), 2.10(s, 9H), 2.06-2.01 (m, 9H), 1.99 (s, 9H), 1.89 (s, 9H), 1.77 (s, 9H),1.52-1.43 (m, 22H), 1.21 (s, 13H), 0.95 (t, 0.1=7.2 Hz, 1H), 0.80 (s,9H), −0.013 (s, 3H), −0.046 (s, 3H).

Synthesis of Compound 146: To a solution of compound 145 (103 mg, 0.0347mmol) in CH₃CN (5 mL) were added HBTU (26 mg, 0.0694 mmol), iPr₂NEt(0.026 mL, 0.149 mmol) and CPG-NH₂ (Prime Synthesis CPG-500, NH₂ loadingconsidered as 80 μmol/g) (450 mg, 0.036 mmol). The mixture was shakenfor 24 hours, then filtered, washed with CH₂Cl₂, and dried in vacuo. Theresidual amino groups were capped by shaking for 1 hour with pyridine(7.5 mL), acetic anhydride (2.5 mL) and triethylamine (0.5 mL).Filtering, washing with CH₂Cl₂ (100 mL), then 50% MeOH/CH₂Cl₂ (100 mL),and drying in vacuo afforded compound 146. Loading: 49 μmol/g.

Example 61: Primary Hepatocyte Binding for Triantennary and 1+1+1 LigandDesigns

siRNA-Ligand conjugates were prepared. The siRNA in each conjugate wasthe same and targeted to TTR. The following ligands were attached to the3′ end of the sense strand of each siRNA. The structure of the ligandson the conjugates was the same as on conjugate 43527, except for thereplacement of the sugar groups as indicated below,

FIG. 13 shows the primary hepatocyte binding affinities for siRNA-ligandconjugates 61696, 61695, 61692, 61694, 61697, 61693, 43527 and 61698,the structures of which are shown below. Binding affinity Ki values arepresented in the table below.

Std. Conjugate Ligand Ki (nM) Error 61696 Tri-α-C6- 2.2 0.2 Azido 61695Tri-β-C6- 3.5 0.4 Azido 61692 Tri-α-C6- 4.8 0.8 Bicyclo 61694 Tri-β-C6-5.2 0.8 Anisole 61697 1 + 1 + 1-β- 9.8 1.4 C6-Anisole 61693 Tri-α-C6-10.8 1.7 Anisole 43527 Triantennary- 15.9 3.4 Parent 61698 1 + 1 + 1-α-19.8 2.9 C6-Anisole

Example 62: SiRNA GalNAC Conjugates with High Affinity Ligands

siRNA-Ligand conjugates were prepared. The siRNA in each conjugate wasthe same and targeted to TTR. The following ligands were attached to the3′ end of the sense strand of each siRNA. The structure of the ligandson the conjugates was the same as on conjugate 57727, except for thereplacement of the sugar groups as indicated below. The structures ofL224, L223, L221, L96 and L227 are provided below.

Conjugate Ligand Ligand type 57727 L96 GalNAc 63189 L223 β-Azido 63190L224 α-Azido 63191 L221 β-Anisole

FIGS. 3A and 3B show the circulating serum mTTR SiRNA levels after 72hours (FIG. 3A) and 144 hours (FIG. 3B) following a single subcutaneousdose of conjugates 57727, 63189, 63192, 63190 and 63191 to miceaccording to the protocol in Example 33.

Example 63: Compounds for T-2′-GalNAc Building Block

Structures

Example 64: Compounds for T-3′-GalNAc Building Block

Structures

Example 65

2′- and 3′-O-phthalimidohexyl-5-methyluridine (2A, 2B).2′,3′-O-dibutylstannylene-5-methyluridine (2.0 g, 4.1 mmol) wassuspended in DMF (10 mL). 6-bromohexyl phthalimide (2.5 g, 8.2 mmol) andNaI (120 mg, 0.82 mmol) were added to the suspension. The reagents weremicrowaved for 3.5 hours at 100° C. resulting in a dark brown homogenousmixture. DMF was evaporated in vacuo and the residue adsorbed to silicagel. The silica gel was loaded into a cartridge for silica gelchromatography. The 2′- and 3′-isomers of theO-phthalimidohexyl-5-methyuridine eluted as an inseparable mixture toyield 890 mg of 2A and 2B (1.8 mmol, 45%).

¹H NMR (400 MHz, DMSO-d₆) δ 11.29 (s, 1H, D₂O exchangeable), 7.88-7.80(m, 4H), 7.78-7.76 Om 1H), 7.73-7.70 (m 0H), 5.81 (d, J=5.3 Hz, 1H),5.72 (d, J=5.6 Hz, 1H), 5.25 (d, J=6.2 Hz, 1H, D₂O exchangeable), 5.12(t, J=5.0 Hz, 1H, D20 exchangeable), 5.00 (d, J=5.9 Hz, 1H, D20exchangeable), 4.14 (q, J=5.6 Hz, 1H), 4.07 (q, J=5.0 Hz, 1H), 3.90-3.79(m, 2H), 3.74 (t, J=4.6 Hz, 0H), 3.67-3.58 (m, 1H), 3.58-3.49 (m, 4H),3.46-3.37 (m, 1H), 1.75 (d, J=3.7 Hz, 3H), 1.62-1.42 (m, 4H), 1.37-1.20(m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 168.00, 167.97, 163.78, 163.73,150.79, 150.57, 136.23, 136.07, 134.44, 134.40, 131.60, 131.56, 123.02,109.38, 109.26, 87.73, 85.89, 85.07, 82.71, 80.82, 77.49, 72.43, 69.69,69.51, 68.38, 60.86, 60.62, 29.23, 28.96, 27.93, 26.15, 26.07, 25.16,24.96, 12.26. MS calculated for C₂₄H₂₉N₃O₈ 487.1955, found m/z 488.0(M+1)⁺, 510.2 (M+23)^(Na+), 486.2 (M−1)⁻, 522.2 (M+35)^(Cl—), R_(f)=0.26in 5% MeOH/DCM v/v

5′-O-Dimethoxytrityl-2′-O-phthalimidohexyl-5-methyluridine (3A). Themixture of 2A and 2B (890 mg, 1.83 mmol) was co-evaporated with pyridineand then dissolved in pyridine (10 mL) under an argon atmosphere andcooled to 0° C. in an ice bath. To this mixture, DMTrCl (690 mg, 2.04mmol) was added, and the reaction was stirred overnight while allowed towarm to room temperature. An additional 0.55 eq of DMTrCl was added andthe reaction was stirred an additional 2 hours. The reaction wasquenched with MeOH and evaporated in vacuo. The crude 2′ and 3′ isomers(3A and 3B) were dissolved in DCM and the organic layer washed twicewith brine. The organic layer was dried with Na₂SO₄ and evaporated invacuo. The compounds were purified via silica gel chromatography andconcentrated in vacuo to yield 510 mg of 3A (0.65 mmol, 35%). The2′-O-alkylated isomer was characterized by identification of the 3′-OHby D₂O exchange followed by COSY.

¹H NMR (400 MHz, DMSO-d₆) δ 11.35 (s, 1H, D₂O exchangeable), 7.88-7.77(m, 4H), 7.48 (s, 1H), 7.38 (d, J=7.4 Hz, 2H), 7.33-7.19 (m, 7H), 6.89(d, J=8.0 Hz, 4H), 5.82 (d, J=4.8 Hz, 1H), 5.10 (d, J=6.3 Hz, 1H, D₂Oexchangeable), 4.18 (q, J=5.5 Hz, 1H), 3.96 (q, J=4.8, 4.4 Hz, 2H), 3.72(s, 6H), 3.62-3.46 (m, 4H), 3.27-3.14 (m, 2H), 1.59-1.45 (m, 4H), 1.38(s, 3H), 1.34-1.20 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.91, 163.61,158.19, 158.16, 150.41, 144.66, 135.45, 135.31, 135.11, 134.33, 131.58,129.74, 127.93, 127.65, 126.84, 122.97, 113.27, 109.60, 86.46, 85.91,83.09, 80.53, 69.63, 68.76, 63.19, 55.06, 37.31, 28.89, 27.91, 26.07,24.95, 11.66. MS calculated for C₄₅H₄₇N₃O₁₀ 789.3261, found m/z 812.3(M+23)^(Na+), 788.3 (M−1)⁻. 824.3 (M+35)^(Cl−) R_(f)=0.35 in 60%EtOAc/Hexanes v/v

5′-O-Dimethoxytrityl-3′-O-phthalimidohexyl-5-methyluridine (3B). The3′-isomer (3B) was separated from the 2′-isomer (3A) during silica gelchromatography and concentrated ill vacuo to yield 420 mg of 3B (29%,0.53 mmol). The 3′-O-alkylated isomer was characterized in the samemanner but with identification of the 2′-OH by D₂O exchange.

¹H, NMR (400 MHz, DMSO-d₆) δ 11.34 (s, 1H, D₂O exchangeable), 7.87-7.79(m, 4H), 7.49 (s, 1H), 7.36 (d, J=7.4 Hz, 2H), 7.29 (t, J=7.6 Hz, 2H),7.26-7.19 (m, 5H), 6.94-6.81 (m, 4H), 5.71 (d, J=4.6 Hz, 1H), 5.36 (d,J=6.0 Hz, 1H, D₂O exchangeable), 4.27 (q, J=5.2 Hz, 1H), 3.99-3.95 (m,1H), 3.90 (t, J=5.3 Hz, 1H), 3.71 (s, 6H), 3.63-3.47 (m, 3H), 3.36 (t,J=6.8 Hz, 1H), 3.26-3.15 (m, 2H), 1.59-1.39 (m, 7H), 1.28-1.20 (m, 4H).¹¹C NMR (100 MHz, DMSO-d₆) δ 167.93, 163.69, 158.17, 158.15, 150.57,144.63, 135.72, 135.30, 135.17, 134.35, 131.58, 129.71, 127.91, 127.63,126.82, 122.97, 113.24, 109.37, 88.69, 85.91, 80.60, 77.27, 72.18,69.67, 55.03, 39.50, 37.33, 29.09, 27.88, 26.08, 25.08, 11.74. MScalculated for C-₄₅H₄₇N₃O₁₀ 789.3261, found m/z 812.0 (M+23)^(Na+),788.3 (M−1)⁻, 824.3 (M+35)^(Cl−) R_(f)=0.18 in 60% EtOAc/Hexanes v/v.

5′-O-Dimethoxytrityl-2′-O-aminohexyl-5-methyluridine (4A*). To asolution of 3A (obtained from RI Chemicals, lot #111010-04, 15.0 g,18.99 mmol) in WON (190 mL) was added hydrazine (3.04 g, 94.52 mmol) andthe heterogeneous mixture was heated to reflux for 3.5 hours. Themixture was cooled to room temperature and evaporated in vacuo to yielda white powder. The product was dissolved in DCM and washed withammonium hydroxide. Brine was added to help remove the emulsion. Theorganic layer was dried with MgSO₄ and evaporated in vacuo to yield11.80 g of crude product which was used without purification for thenext step.

MS calculated for C₃₇H₄₅N₃O₈ 659.3207, found m/z 660.2 (M+1)⁺, 682.1(M+23)^(Na+), 658.1 (M−1)⁻, 694.1 (M+35)^(Cl−), R_(f)=0.02 in 5%MeOH/DCM v/v. ¹H NMR (400 MHz, DMSO-d₆) J=7.48 (s, 1H), 7.38 (d, J=7.5Hz, 2H), 7.30 (t, J=7.5 Hz, 2H), 7.24 (d, J=8.9 Hz, 5H), 6.89 (d, J=8.4Hz, 4H), 5.84 (d, J=5.0 Hz, 1H), 5.74 (s, 1H), 4.19 (t, J=5.0 Hz, 1H),3.97 (t, J=4.9 Hz, 2H), 3.72 (s, 6H), 3.63-3.45 (m, 3H), 3.27-3.15 (m,3H), 1.48 (d, J=6.4 Hz, 2H), 1.38 (s, 3H), 1.34-1.18 (m, 6H).

5′-O-Dimethoxytrityl-r-O-aminohexyl-C5-GalNAc(0-Bz)-5-methyluridine(4A). To a solution of crude 4A* (6.00 g, 9.09 mmol) in DCM (250 mL) wasadded triethylamine (3.8 mL, 27.30 mmol) and the mixture was allowed tostir for 10 minutes. GalNAc-C5-NHS ester (7.31 g, 10.00 mmol) was addedand the reaction mixture was stirred for 2 hours. The reaction mixturewas washed with saturated bicarbonate and the organic layer was driedwith Na₂SO₄ then evaporated in vacuo. The crude product was purified viasilica gel chromatography to yield 9.56 g of 4A (7.49 mmol, 82%).

¹H NMR (400 MHz, DMSO-d₆) δ 11.37 (s, 1H, D₂O exchangeable), 7.97 (d,J=9.3 Hz, 1H, D₂O exchangeable), 7.91 (t, J=6.8 Hz, 4H), 7.73-7.45 (m,11H), 7.41-7.19 (m, 11H), 6.88 (d, J=8.4 Hz, 4H), 5.84 (d, J=4.7 Hz,1H), 5.74 (d, J=3.4 Hz, 1H), 5.36 (dd, J=11.1, 3.3 Hz, 1H), 5.12 (d,J=6.3 Hz, 1H, D₂O exchangeable), 4.73 (d, J=8.5 Hz, 1H), 4.45 (q, J=8.8,7.6 Hz, 2H), 4.39-4.15 (m, 3H), 3.96 (1. J=4.7 Hz, 2H), 3.79 (dd, J=9.4,3.8 Hz, 1/1), 3.72 (s, 6H), 3.63-3.45 (m, 3H), 3.28-3.16 (m, 2H), 2.99(q, J=65 Hz, 2H), 2.04 (s, 2H), 1.69 (s, 3H), 1.55-1.43 (m, 6H), 1.36(d, J=17.6 Hz, 5H), 1.24 (s, 4H), NMR (100 MHz, DMSO-d₆) δ 171.73,169.40, 165.20, 165.16, 164.86, 163.62, 158.18, 158.15, 150.41, 144.64,135.47, 135.31, 135.10, 133.77, 133.49, 129.73, 129.20, 129.16, 129.03,129.00, 128.97, 128.70, 128.59, 127.91, 127.64, 126.82, 113.26, 109.59,100.89, 86.47, 85.91, 83.09, 80.60, 71.85, 69.97, 69.74, 68.77, 67.92,63.19, 62.03, 55.04, 49.74, 38.34, 35.03, 29.17, 29.04, 28.59, 26.25,25.12, 22.69, 21.85, 11.66. MS calculated for C₇₁H₇₈N₄O₁₈ 1274.5311,found m/z 1298.3 (M+23)^(Na+), 1309.4 (M+35)^(Cl−). R_(f)=0.36 in 5%MeOH/DCM v/v.

5′-O-Dimethoxytrityl-2′-O-aminohexyl-C5-GalNAc(0-Bz)-3′43-succinate-5-methyluridine(5A). To a solution of 4A (2.00 g, 1.57 mmol) in DCM (50 mL) was addedDMAP (574 mg, 4.70 mmol) and succinic anhydride (313 mg, 3.14 mmol). Thereaction mixture was stirred over night at room temperature. The productwas purified via silica gel chromatography (ϕ=4.2 cm×15 cm, pretreatedwith 2% TEA in DCM). The product was eluted with 0-5% MeOH and 2-5% Et₃Nin DCM v/v and co-evaporated with acetonitrile in vacuo to yield 2.11 g(1.43 mmol, 91%) of 6a as an Et₃N salt.

¹H NMR (400 MHz, DMSO-d₆) δ 11.44 (s, 1H), 8.05 (d, J=9.3 Hz, 1H), 7.91(t, J=6.8 Hz, 4H), 7.74-7.45 (m, 11H), 7.40-7.27 (m, 6H), 7.23 (d, J=8.7Hz, 5H), 6.89 (d, J=8.1 Hz, 4H), 5.84 (d, J=6.1 Hz, 1H), 5.74 (d, J=3.5Hz, 1H), 5.36 (dd, J=11.1, 3.3 Hz, 1H), 5.27-5.22 (m, 1H), 4.74 (d,J=8.5 Hz, 1H), 4.48-4.40 (m, 2H), 4.38-4.22 (m, 3H), 4.13 (q, J=3.5 Hz,1H), 3.78 (d, J=9.7 Hz, 1H), 3.72 (s, 6H), 3.54-3.28 (m, 5H), 3.25-3.19(m, 1H) 2.97 (q. J=6.5 Hz, 2H), 2.57-2.51 (m, 2H), 2.44 (t, J=6.5 Hz,2H), 2.04 (s, 2H), 1.69 (s, 3H), 1.57-1.27 (m, 11H), 1.18 (s, 4H), ¹³CNMR (100 MHz, DMSO-d₆) δ 173.40, 171.76, 171.63, 169.39, 165.20, 165.15,164.86, 163.50, 158.22, 150.50, 144.48, 135.45, 135.12, 134.95, 133.76,133.48, 129.70, 129.17, 129.03, 129.00, 128.97, 128.70, 128.58, 127.95,127.61, 113.30, 110.16, 100.91, 86.13, 80.75, 78.22, 71.89, 70.67,70.23, 69.97, 68.73, 67.91, 62.04, 55.04, 52.01, 49.73, 38.33, 34.98,29.13, 28.92, 28.55, 26.19, 25.08, 22.68, 21.82, 11.69, 10.48. MScalculated for C-₇₅H₈₁N₄O₂₁ 1374.5472. found m/z 1397.4 (M+23)^(Na+),1373.4 (M−1)⁻, 1409.4 (M+35)^(Cl−). R_(f)=0.41 in 5% MeOH/5% Et₃N/DCMv/v

5′-O-Dimethoxytrityl-2′43-aminohexyl-C5-GalNAc(0-Bz)-3′-O-CPG-5-methyluridine(6A). To a solution of 5A (2.01 g, L36 mmol) in acetonitrile (100 mL)was added HBTU (1.03 g, 2.72 mmol) and DIEA (528 mg, 4.08 mmol). Themixture was shaken for 5 minutes before the addition of CPG (16.00 g,130 μmol/g, 540 Å). The mixture was shaken for 24 hours. CPG wasfiltered and washed with DCM. 20% MeOH in DCM v/v, then ether. CPG wasevaporated in vacuo and then treated with acetic anhydride (25 mL) inpyridine (75 mL) and Et₃N (1 mL) and shaken for 1 hour. CPG was filteredand washed with the same solvents as described above. The averageloading was determined by trityl absorbance spectroscopy measurements oftwo samples and was calculated to be 73 μmol/g.

5′-O-Dimethoxytrityl-2′-O-aminohexyl-C5-GalNAc(O-Bz)-3′-O—(N,N-diisopropyl)-β-cyanoethylphosphoramidite-5-methyluridine(7A). 4A (2.90 g, 2.27 mmol) was co-evaporated with anhydrousacetonitrile twice then kept under a strict argon atmosphere. To asolution of 4A in anhydrous DCM (35 mL) at 0° C. was added2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite (1.37 g, 4.55mmol) followed by DCI (268 mg, 2.27 mmol). The mixture was stirred at 0°C. for 20 minutes then at room temperature for 17 hours. The product waswashed with saturated bicarbonate and extracted with DCM. The organiclayer was dried with Na₂SO₄ to yield a pale yellow foam. Silica gelchromatography (ϕ=4.2 cm×19 cm pretreated with 50% EtOAc and 1% TEA inhexane) was carried out. The column was washed with 80% EtOAc in hexane(8 CV) followed by 100% EtOAc (8 CV), then 3% MeOH in DCM (5 CV).Product 7A eluted with 100% EtOAc and again in 3% MeOH. Fractionscontaining 7A were combined and evaporated in vacuo to yield 3.20 g of7A (2.17 mmol, 95%).

¹H NMR (400 MHz, DMSO-d₆) δ 11.39 (s, 1H), 7.98 (d, J=9.3 Hz, 1H), 7.91(1, J=7.2 Hz, 4H), 7.72-7.45 (m, 11H), 7.38 (t, J=6.8 Hz, 4H), 7.33-7.20(m, 7H), 6.88 (t, J=5.4 Hz, 4H), 5.82 (d, J=4.3 Hz, 1H), 5.75 (s, 1H),5.35 (dd, J=11.1, 3.1 Hz, 1H), 4.73 (d, J=8.5 Hz, 1H), 4.48-4.31 (m,4H), 4.15-4.04 (m, 2H), 3.71 (s, 8H), 3.58-3.45 (m, 5H), 3.28-3.20 (m,2H), 3.03-2.93 (m, 2H), 2.76 (t, J=5.8 Hz, 1H), 2.57 (C₁, J=5.3 Hz, 1H),2.04 (s, 2H), 1.69 (s, 3H), 1.49 (s, 6H), 1.42-1.29 (m, 5H), 1.27-1.15(m, 5H), 1.14-1.03 (m, 10H), 0.94 (d, J 6.7 Hz, 3H). ¹³C NMR (125 MHz,DMSO-d₆) δ 171.60, 169.26, 165.07, 165.03, 164.73, 163.48, 158.10,158.08, 158.07, 150.24, 150.22, 144.39, 144.34, 134.99, 134.97, 134.87,134.80, 133.63, 133.37, 133.34, 129.65, 129.62, 129.60, 129.07, 129.05,129.03, 128.90, 128.88, 128.87, 128.83, 128.56, 128.45, 127.75, 127.56,127.50, 126.74, 118.72, 118.57, 113.10, 113.08, 109.63, 109.54, 100.76,85.92, 85.90, 71.72, 69.84, 68.61, 67.78, 61.89, 54.91, 49.60, 45.53,42.53, 42.42, 42.31, 38.23, 38.21, 34.88, 29.05, 29.02, 28.96, 28.45,26.17, 25.08, 25.04, 24.18, 24.13, 24.09, 24.03, 22.55, 21.71, 19.71,19.66, 19.62, 11.51, 11.49. ³¹P NMR (160 MHz, DMSO-d₆) δ 154.01, 153.65.

MS calculated for C₈₀H₉₅N₆O₁₉P 1474.6390. found m/z 1497.4 (M+23)^(Na+),1509.4 (M+35)^(Cl−). R_(f)=0.39 in 100% EtOAc.

Example 66

5′—O-Dimethoxytrityl-3′-O-aminohexyl-5-methyluridine (4B*). To asolution of 3B (3.64 g, 4.61 mmol ˜1 g) u3 MeOH (46 nil) was addedhydrazine (738 mg, 23.04 mmol) and the reaction mixture was refluxed for5.5 hours. The workup procedure described for 4A* was used to isolatecrude 4B*. Co-evaporation with acetonitrile yielded 2.93 g of crude 4B*.

MS calculated for C₃₇H₄₅N₃O₈ 659.3207. found m/z 660.2 (M+1)⁺, 682.1(M+23)^(Na+), 658.1 (M−1)⁻, 694.1 (M+35)^(Cl−) R_(f)=0.02 in 5% MeOH/DCMv/v

Crude 4B* NMR:

¹H NMR (400 MHz, DMSO-d₆) δ 7.50 (s, 1H), 7.43-7.16 (m, 9H), 6.88 (d,J=8.7 Hz, 4H), 5.73 (d, J=4.6 Hz, 1H), 4.29 (t, J=4.8 Hz, 1H), 4.02-3.89(m, 3H), 3.46-3.15 (m, 5H), 2.24-2.16 (m, 1H), 2.05 (s, 1H), 1.58-1.10(m, 13H), ¹³C NMR (125 MHz, DMSO-d₆) δ 163.73, 158.04, 158.02, 150.58,144.49, 135.54, 135.18, 135.06, 129.56, 127.78, 127.50, 126.69, 113.11,109.23, 88.49, 85.78, 80.47, 77.20, 72.02, 69.62, 62.85, 54.91, 54.90,41.44, 33.12, 29.17, 26.11, 25.31, 11.62.

5′-O-Dimethoxytrityl-3′-O-aminohexyl-C5-GalNAc(O-Bz)-5-methyluridine(4B). To a solution of 4B* (2.85 g, 4.32 mmol) in DCM (45 mL) treatedwith TEA (1.8 mL) was added GalNAc-NHS ester (3.47 g, 4.75 mmol). Thereaction was stirred for 2 hours before an additional 0.2 eq ofGalNAc-NHS ester was added. After 1 hour, the product was isolated inthe same manner as described for 5A. Silica column purification yielded3.62 g of 4B (2.84 mmol).

¹H NMR (400 MHz, DMSO-d₆) δ 11.35 (s, 1H), 8.00-7.89 (m, 5H), 7.73-7.45(m, 11H), 7.40-7.35 (m, 4H), 7.33-7.20 (m, 7H), 6.88 (d, J=8.8 Hz, 4H),5.74 (d, J=10.1 Hz, 2H), 5.42-5.33 (m, 2H), 4.73 (d, J=8.5 Hz, 1H),4.48-4.41 (m, 2H), 4.38-4.23 (m, 3H), 3.99 (d, J=4.7 Hz, 1H), 3.91 (t,J=5.1 Hz, 1H), 3.82-3.76 (m, 1H), 3.72 (s, 6H), 3.62-3.47 (m, 2H),3.41-3.36 (m, 1H), 3.27-3.17 (m, 2H), 2.99 (q, J=6.6 Hz, 2H), 2.04 (t,J=6.4 Hz, 2H), 1.69 (s, 3H), 1.55-1.28 (m, 11H), 1.28-1.15 (m, 4H). ¹³CNMR (75 MHz, DMSO-d₆) δ 171.72, 169.38, 165.19, 165.14, 164.85, 163.64,162.25, 158.14, 150.55, 144.57, 135.68, 135.30, 135.16, 133.73, 133.45,129.66, 129.18, 129.14, 129.00, 128.93, 128.66, 128.55, 127.88, 127.61,126.80, 113.22, 109.36, 100.88, 88.57, 85.90, 80.63, 77.35, 72.16,71.83, 69.97, 69.73, 68.73, 67.91, 62.97, 62.02, 55.01, 54.84, 49.75,38.34, 35.73, 35.02, 30.73, 29.23, 29.11, 28.57, 26.25, 25.21, 22.66,21.84, 11.68. MS calculated for C₇₁H₇₈N₄O₁₈ 1274.5311, found m/z 1297.4(M+23)^(Na+). 1309.4 (M+35)^(Cl−). R_(f)=0.24 in 5% MeOH in DCM).

5′-O-Dimethoxytrityl-3′-O-aminohexyl-C5-GalNAc(O-Bz)-2′-O-succinate-5-methyluridine(5B). To a solution of 4B (1.10 g, 0.86 mmol) in DCM (20 mL) and DMAP(315 mg, 2.58 mmol) was added succinic anhydride (172 mg, 1.73 mmol).The reaction mixture was stirred for 23 hours then purified using theprocedure described for 5A and co-evaporated with acetonitrile in vacuoto yield 1.03 g of 5B (0.70 mmol, 81%).

¹H NMR (400 MHz, DMSO-d₆) δ 8.09 (d, J=9.3 Hz, 1H), 7.91 (t, J=6.9 Hz,4H), 7.76 (t, J=5.4 Hz, 1H), 7.73-7.51 (m, 9H), 7.47 (t, J=7.7 Hz, 2H),7.40-7.19 (m, 12H), 6.87 (d, J=8.7 Hz, 4H), 5.85 (d, J=3.9 Hz, 1H), 5.74(d, J=3.2 Hz, 1H), 5.47-5.42 Om 1H), 5.36 (dd, J=11.1, 3.2 Hz, 1H), 4.75(d, J=8.5 Hz, 1H), 4.44 (q, J=9.1, 7.7 Hz, 2H), 4.38-4.20 (m, 4H),4.01-3.94 (m, 2H), 3.80-3.76 (m, 2H), 3.71 (s, 6H), 3.40-3.16 (m, 10H),3.07-2.94 (m, 3H), 2.56 (q, J=6.2, 5.7 Hz, 2H), 2.43 (d, J=4.1 Hz, 3H),2.07-2.01 (m, 2H), 1.69 (s, 3H), 1.48 (d, J=15.0 Hz, 7H), 1.40-1.27 (m,4H), 1.16 (s, 4H), ¹³C NMR (100 MHz, DMSO-d₆) δ 173.63, 173.45, 172.79,171.92, 171.52, 169.54, 168.92, 165.29, 165.24, 164.94, 163.78, 158.23,150.33, 144.57, 136.18, 135.27, 135.18, 133.84, 133.57, 129.77, 129.24,129.09, 129.06, 129.04, 128.77, 128.65, 127.96, 127.70, 126.90, 113.29,109.73, 100.97, 85.97, 80.75, 75.82, 73.31, 71.96, 70.48, 62.59, 62.40,55.10, 55.07, 52.06, 51.40, 51.36, 45.45, 38.42, 35.04, 33.35, 29.20,29.09, 28.95, 28.77, 22.70, 21.90, 11.81, 10.08, 7.26, 7.17. MScalculated for C₇₅H₈₁N₄O₂₁ 1374.5472, found m/z 1397.4 (M+23)^(Na+),1373.4 (M−1)⁻. R_(f)=0.18 in 5% MeOH in DCM).

5′-O-Dimethoxytrityl-3′-O-aminohexyl-C5-GalNAc(0-Bz)-2′43-CPG-5-methyluridine(6B). To a solution of SB (970 mg, 0.66 mmol) in acetonitrile (50 mL)was added HBTU (497 mg, 1.31 mmol) and DIEA (339 mg, 1.97 mmol). After 5minutes of shaking, CPU (8.20 g, 130 μmol/g, 540 Å) was added andshaking was continued for 21 hours. The CPG was removed by Filtration,washed, capped, and loading determined as described for 6A to yield CPGwith an average loading of 56 mol/g.

5′-O-Dimethoxytrityl-3′-O-aminohexyl-C5-GalNAc(O-Bz)-2′-O-(cyanoethyl-N,N-diisopropyl)-phosphoramidite-5-methyluridine(7B). 4B (1.98 g, 1.55 mmol) was prepared in the same manner describedin 7A and treated with the same reagents. The product 7B was loaded ontoa column prepared in the same manner as described for 7A and eluted with70% EtOAc in hexane (4 CV), 80% EtOAc in hexane (10 CV), 100% EtOAc (2CV), then 3% MeOH in DCM (4 CV). The desired product eluted in 100%EtOAc and in 3% MeOH. Fractions containing the desired product werecombined and evaporated in vacuo to yield 1.89 g (1.28 mmol, 83%) of 7B.

¹H NMR (400 MHz, DMSO-d₆) δ 11.38 (s, 1H), 8.00-7.88 (m, 5H), 7.72-7.45(m, 11H), 7.41-7.34 (m, 4H), 7.33-7.20 (m, 7H), 6.91-6.85 (m, 4H), 5.88(dd, J=9.3, 5.1 Hz, 1H), 5.75 (d, J=3.3 Hz, 1H), 5.36 (dd, J=11.1, 3.3Hz, 1H), 4.73 (d, J=8.5 Hz, 1H), 4.62-4.50 (m, 1H), 4.45 (q, J=8.2, 7.2Hz, 2H), 4.38-4.23 (m, 2H), 4.07-3.95 (m, 3H), 3.72 (s, 13H), 3.44-3.39(m, 1H), 3.31-3.22 (m, 2H), 2.98 (s, 2H), 2.71-2.66 (m, 1H), 2.04 (s,2H), 1.69 (s, 3H), 1.47 (d, J=28.1 Hz, 8H), 1.40-1.18 (m, 8H), 1.13-1.00(m, 11H), ¹³C NMR (125 MHz, DMSO-d₆) δ 171.72, 171.70, 170.37, 170.29,169.37, 165.18, 165.14, 164.85, 163.58, 163.50, 158.20, 158.18, 158.16,158.14, 154.86, 150.48, 150.43, 144.53, 144.52, 144.50, 135.18, 135.14,135.05, 134.98, 133.74, 133.48, 133.44, 129.69, 129.66, 129.63, 129.18,129.16, 129.13, 129.01, 128.99, 128.98, 128.93, 128.67, 128.64, 128.56,128.54, 128.52, 127.91, 127.88, 127.57, 118.81, 118.70, 113.25, 113.21,109.72, 109.68, 100.87, 86.16, 86.15, 86.02, 71.83, 71.81, 69.95, 69.92,68.72, 68.71, 67.89, 63.45, 59.71, 55.00, 54.98, 49.72, 42.79, 42.69,42.64, 38.35, 38.33, 35.00, 30.12, 30.03, 29.26, 29.25, 29.14, 29.11,29.09, 28.56, 28.54, 26.29, 25.27, 25.25, 24.34, 24.29, 24.24, 24.22,24.08, 24.02, 22.66, 22.65, 21.84, 21.83, 21.36, 20.72, 20.67, 20.66,19.80, 19.78, 19.74, 19.73, 19.67, 19.62, 18.82, 18.56, 16.57, 14.04,13.65, 13.50, 11.69, 11.56, 11.56, 11.55, 11.53. ³¹P NMR. (162 MHz,DMSO-d₆) δ 155.08, 154.60. MS calculated for C₈₀H₉₅N₆O₁₉P 1474.6390,found m/z 1497.4 (M+23)^(Na+), 1474.3 (M−1)⁻, 1509.4 (M+35)^(Cl−)R_(f)=0.43 in 100% EtOAc).

Example 67

LRMS calculated for C₂₄H₂₈N₆O₆ 496.52. found m/z 497.2 (M+1)⁺519.2(M+23)^(Na+), 486.2 (M−1)⁻, 522.2 (M+35)^(Cl−).

(2A/2B)—Adenosine (20 g, 74.8 mmol) was treated with NaH (4.5 g, 112mmol) in DMF (200 ml) at 0° C. for 20 minutes. N-(Iodohexyl)phthalimide(30.7 g, 86 mmol) was added to the solution and then heated to 80° C.for two days. DMF was evaporated in vacuo to yield a pale orange gumcontaining the and 3′-O-alkylated isomers. The crude mixture wasadsorbed onto silica gel and purified (5% MeOH/DCM v/v) to yield 5.1 g(10.3 mmol, 14%) of 2A as well as a mixture of the regioisomers.

¹H NMR (400 MHz, DMSO-d₆) δ 8.36 (s, 1H), 8.12 (s, 1H), 7.95-7.68 (m,4H), 7.32 (s, 2H, D₂O exchangeable), 5.96 (d, J=6.2 Hz, 1H), 5.42 (t,J=7.1, 4.6 Hz, 1H, D₂O exchangeable), 5.15 (d, J=5.1 Hz, 1H, D₂Oexchangeable), 4.44 (t, J=6.3, 4.7 Hz, 1H), 4.27 (q, J=4.9, 2.8 Hz, 1H),3.96 (q, J=3.4 Hz, 1H), 3.66 (dt, J=12.2, 4.2 Hz, 1H), 3.60-3.42 (m,4H), 3.30 (dd, J=9.5, 6.4 Hz, 1H), 1.41 (dt, J=33.8, 6.9 Hz, 4H),1.25-1.04 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.89, 150.68, 148.24,148.19, 134.35, 131.54, 128.38, 127.61, 125.44, 122.96, 118.74, 86.39,86.03, 81.49, 69.66, 68.64, 28.85, 27.81, 25.93, 24.84. LRMS calculatedfor C-₂₄H₂₈N₆O₆ 496.2070. found m/z 497.2 (M+1)⁺, 519.2 (M+23)^(Na+),531.2 (M+35)^(Cl−) R_(f)=0.28 in 5% MeOH/DCM v/v.

(3A)—Compound 2A (7.4 g, 14.9 mmol) was coevaporated with pyridine thendissolved in pyridine (75 ml) under argon gas. Triethylamine (3.2 ml,22.4 mmol) and 4-dimethylaminopyridine (45 mg, 0.37 mmol) were added andthe reaction mixtured was stirred for 15 minutes. DMTrCl (5.6 g, 16.4mmol) was then added and stirred overnight. An additional 1.5 g ofDMTrCl was then added to drive the reaction to completion. The reactionmixture was quenched with MeOH (5 ml) and evaporated in vacuo. The crudeproduct was washed with saturated NaHCO₃, extracted with EtOAc and driedwith Na₂SO₄. The crude product was purified with a silica column (2.5%MeOH/DCM v/v) to yield 7.8 g (13 mmol, 66%) of pure 3A.

¹H NMR (400 MHz, DMSO-d₆) δ 8.26 (s, 1H), 8.09 (s, 1H), 7.88-7.77 (m,4H), 7.42-7.33 (m, 2H), 7.33-7.16 (m, 9H), 6.88-6.78 (m, 4H), 6.01 (d,J=4.8 Hz, 1H), 5.17 (d, J=5.9 Hz, 1H), 4.56 (1, J=5.0 Hz, 1H), 4.38 (q,J=5.2 Hz, 1H), 4.06 (q, J=4.6 Hz, 1H), 3.72 (s, 6H), 3.62-3.37 (m, 4H),3.34 (s, 2H), 3.23 (d, J=4.6 Hz, 2H), 1.60-1.37 (m, 4H), 1.31-1.11 (m,4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.90, 158.02, 156.08, 152.63,149.21, 144.85, 139.58, 135.55, 135.45, 134.31, 131.57, 129.70, 127.75,127.68, 126.62, 122.95, 119.20, 113.10, 85.97, 85.49, 83.48, 80.10,69.76, 69.12, 63.53, 28.92, 27.85, 25.99, 24.91. LRMS calculated forC-₄₅H₄₆N₆O₈ 798.3377. found m/z 799.2 (M+1)⁺, 821.1 (M+23)^(Na+), 833.1(M+35)^(Cl−) R_(f)=0.33 in 5% MeOH/DCM v/v.

(4A)—Compound 3 (13 g, 16.2 mmol) was dissolved in MeOH (160 ml) andhydrazine (2.6 g, 81 trunol) was added to the solution. The reactionmixture was stirred at reflux for 3 hours. TLC analysis showed completedisappearance of 3A and the appearance of a more polar spot, presumably4A*. The MeOH was evaporated in vacuo and the crude foam was washed withNH₄OH. The aqueous layer was extracted with DCM; saturated NaCl wasneeded to clear the emulsion. The organic layer was dried with MgSO₄ andevaporated in vacuo. 4A* was coevaporated with toluene before beingdissolved in DCM (150 nil) and triethylamine (6.6 ml, 47.6 mmol).GalNAc(OBz)-C5-NHS ester (12.8 g, 17.4 mmol) was added to the mixtureand the reaction proceeded for two hours at room temperature. Anadditional 1.7 g, 2.4 mmol of GalNAc(OBz)-C5-NHS ester was added to thereaction and allowed to stir for an additional two hours. The reactionmixture was evaporated in vacuo and the crude product was washed withNaHCO₃. The aqueous layer was extracted with DCM and dried with Na₂SO₄.Crude 4A was adsorbed to silica and purified (2.5-5% MeOH/DCM v/v) toyield 16.7 g (13.1 mmol, 81%) of 4A.

¹H NMR (400 MHz, DMSO-d₆) δ 8.26 (s, 1H), 8.09 (s, 1H), 8.02-7.85 (m,5H), 7.76-7.45 (m, 10H), 7.44-7.17 (m, 13H), 6.83 (dd, J=8.8, 5.1 Hz,4H), 6.01 (d, J=4.9 Hz, 1H), 5.78-5.74 (m, 1H), 5.37 (dd, J=11, 1, 3.2Hz, 1H), 5.18 (d, J=5.8 Hz, 1H), 4.74 (d, J=8.5 Hz, 1H), 4.57 (t, J=4.9Hz, 1H), 4.46 (q, J=8.6, 7.4 Hz, 2H), 4.41-4.23 (m, 3H), 4.07 (q, J=4.5Hz, 1H), 3.85-3.76 (m, 1H), 3.72 (s, 6H), 3.55 (ddd, J=24.2, 9.5, 5.1Hz, 2H), 3.46-3.38 (m, 1H), 3.23 (d, J=4.4 Hz, 2H), 2.97 (q, J=6.5 Hz,2H), 2.04 (s, 2H), 1.70 (s, 3H), 1.58-1.36 (m, 6H), 1.33-1.12 (m, 6H).¹³C NMR (100 MHz, DMSO-d₆) δ 171.69, 169.38, 165.20, 165.15, 164.85,158.00, 156.07, 152.62, 149.19, 144.82, 139.58, 135.53, 135.42, 133.77,133.51, 133.47, 129.68, 129.19, 129.16, 129.02, 129.00, 128.97, 128.69,128.59, 127.73, 127.65, 126.61, 119.18, 113.08, 100.88, 85.94, 85.47,83.49, 80.08, 71.84, 69.96, 69.80, 69.10, 68.75, 67.91, 63.53, 62.02,54.98, 54.89, 49.73, 35.01, 29.11, 29.03, 28.58, 26.17, 25.05, 22.69,21.83. LRMS calculated for C-₃₇H₄₄N₆O₆ 668.3322. found m/z 691.3(M+23)^(Na+). 703.2 (M+35)^(Cl−) (4A*). LRMS calculated for C₇₁H₇₇N₇O₁₆1283.5427, found m/z 1306.3 (M+23)^(Na+), 1282.3 (M−1)⁻, 1318.3(M+35)^(Cl−) (4A). R_(f)=0.00 in 5% MeOH/DCM v/v (4A*). R_(f)=0.24 in 5%MeOH/DCM v/v (4A)

(5A)—Compound 4A (16 g, 12.5 mmol) was dissolved in DMF (50 ml) andN,N-Dimethylformamide dimethyl acetal (7.4 g, 62.3 mmol) was added tothe stirring solution. The reaction mixture was heated to 60° C. for 3hours before removal of DMF in vacuo. Trace amounts of starting materialwere removed with a silica gel column (2.5% MeOH/DCM v/v) to yield 12.5g (9.3 mmol, 75%) of 5A.

¹H NMR (400 MHz, DMSO-d₆) δ 8.88 (s, 1H), 8.35 (d, J=5.2 Hz, 2H),7.98-7.88 (m, 5H), 7.59 (ddt, J=60.0, 30.9, 7.5 Hz, 11H), 7.37 (dd,J=16.2, 8.2 Hz, 4H), 7.21 (q, J=7.3 Hz, 8H), 6.81 (dd, J=8.8, 7.2 Hz,4H), 6.05 (d, J=5.0 Hz, 1H), 5.74 (d, =3.3 Hz, 1H), 5.35 (dd, J=11.1,3.3 Hz, 1H), 5.18 (d, J=5.9 Hz, 1H), 4.72 (d, J=8.5 Hz, 1H), 4.58 (t,J=5.0 Hz, 1H), 4.44 (q, J=8.3, 7.3 Hz, 2H), 4.39-4.22 (m, 3H), 4.06 (q,J=4.6 Hz, 1H), 3.78 (dd, J=9.9, 4.5 Hz, 1H), 3.71 (s, 6H), 3.53 (dtd,J=19.4, 9.5, 5.1 Hz, 2H), 3.40 (dt, J=9.4, 6.4 Hz, 1H), 3.22 (d, J=4.5Hz, 2H), 3.18 (s, 3H), 3.11 (s, 3H), 2.93 (q, J=6.5 Hz, 2H), 2.02 (s,2H), 1.68 (s, 3H), 1.53-1.36 (m, 7H), 1.30-1.08 (m, 7H). ¹³C NMR (125MHz, DMSO-d₆) δ 171.68, 169.38, 165.19, 165.15, 164.85, 159.23, 158.01,157.98, 157.91, 151.93, 151.19, 144.80, 141.39, 135.49, 135.44, 133.76,133.49, 133.46, 129.68, 129.63, 129.18, 129.17, 129.15, 129.02, 129.00,128.99, 128.96, 128.68, 128.58, 127.73, 127.65, 126.61, 125.76, 118.03,100.88, 85.97, 85.47, 83.59, 80.01, 71.84, 69.96, 69.79, 69.11, 68.74,67.90, 63.54, 62.51, 62.02, 54.97, 54.95, 54.88, 51.97, 49.72, 45.62,35.00, 34.52, 29.07, 29.00, 28.57, 26.14, 25.03, 22.68, 21.83, 7.15.LRMS calculated for C₇₄H₈₂N₈O₁₆ 1338.5849, found m/z 1339.4 (M), 1361.4(M+23)^(Na+), 1338.4 (M−1)⁻, 1373.4 (M+35)^(Cl−). R_(f)=0.29 in 5%MeOH/DCM v/v.

(6A)—Compound 5A (2 g, 1.5 mmol) was dissolved in DCM (15 ml) and4-Dimethylaminopyridine (550 mg, 4.5 mmol) was added to the stirringmixture. Succinic anhydride (300 mg, 3 mmol) was added and the solutionwas stirred at room temperature for 3 hours. DCM was evaporated in vacuoand the crude foam was loaded onto a 2% triethylamine in DCM v/vpretreated manual column (ϕ=4.6×17). A gradient of 1-5% MeOH/2-5%triethylamine/DCM v/v was used to purify 6A. 6A came at 3% MeOH/3%triethylamine/DCM v/v in quantitative yield.

¹H NMR (500 MHz, DMSO-d₆) δ 8.89 (s, 1H), 8.42 (s, 1H), 8.30 (s, 1H),8.09 (d, J=9.3 Hz, 1H), 7.91 (t, J=8.2 Hz, 4H), 7.75-7.65 (m, 4H),7.65-7.53 (m, 4H), 7.47 (t, J=7.6 Hz, 2H), 7.37 (dd, J=13.9, 7.3 Hz,4H), 7.21 (td, J=12.2, 10.6, 5.5 Hz, 7H), 6.82 (t, J=8.3 Hz, 4H), 6.04(d, J=6.6 Hz, 1H), 5.74 (d, J=3.1 Hz, 1H), 5.45-5.41 (m, 1H), 5.36 (dd,J=11.1, 3.2 Hz, 1H), 5.08-5.02 (m, 1H), 4.75 (d, J=8.5 Hz, 1H),4.48-4.39 (m, 3H), 4.38-4.19 (m, 5H), 3.78 (d, J=9.4 Hz, 2H), 3.70 (s,6H), 3.50 (d, J, 9.3 Hz, 2H), 3.40-3.26 (m, 6H), 3.18 (s, 3H), 3.11 (s,3H), 2.59 (q, J=6.8, 6.3 Hz, 2H), 2.03 (s, 2H), 1.69 (s, 3H), 1.49 (s,4H), 1.34-1.16 (m, 6H), 1.08-0.97 (m, 4H), ¹³C NMR (125 MHz, DMSO-d₆) δ173.35, 171.73, 171.48, 169.39, 165.20, 165.14, 164.85, 159.33, 158.06,158.03, 157.96, 151.94, 151.22, 144.68, 141.81, 135.31, 133.75, 133.48,133.45, 129.69, 129.61, 129.17, 129.16, 129.14, 129.01, 128.98, 128.95,128.68, 128.57, 127.75, 127.61, 126.66, 125.86, 113.11, 100.88, 85.82,85.69, 81.39, 77.45, 71.89, 71.01, 70.19, 69.96, 68.70, 67.89, 63.28,62.04, 54.98, 54.96, 52.01, 49.71, 34.95, 34.54, 29.00, 28.92, 28.85,28.81, 28.53, 26.05, 24.95, 22.67, 21.80, 7.18. LRMS calculated forC₇₈H₈₆N₈O₁₉ 1438.6009. found m/z 1439.4 (M), 1463.4 (M+23)^(Na+), 1437.4(M−1)⁻. R_(f)=0.23 in 5% MeOH/5% Et₃N/DCM v/v

(7A)—Compound 6A (2.2 g, 1.4 mmol) was dissolved in acetonitrile (110ml) and HBTU (1.1 g, 2.9 mmol) and DIEA (550 mg, 4.3 mmol) were added.The mixture was shaken for 5 minutes then LCAA-CPG (18 g, 540A, 130μmol/g) was added and shaken over night at room temperature. The CPG wasfiltered and washed with 300 ml each of DCM, 20% MeOH/DCM v/v, anddiethyl ether then dried in vacuo. The CPG was shaken for 1 hour inacetic anhydride (25 ml), pyridine (75 ml), and triethylamine (1 ml)before being washed again by the same conditions as before. Compound 7Awas dried in vacuo overnight and loading was measured with aspectrophotometer (72 μmol/g).

(8A)—Compound 5A (1.0 g, 0.75 mmol) was coevaporated with ACN twice andput under a strict argon atmosphere. DCM (7.5 ml) was added to the flaskand cooled to 0° C. before the addition of 2-CyanoethylN,N,N′,N′-tetraisopropylphosphordiamidite (450 mg, 1.5 mmol). Themixture was stirred for 20 minutes then 4,5-Dicyanoimidazole (90 mg,0.75 mmol) was added to the reaction. The reaction was slowly warmed toroom temperature over night. The reaction was washed with saturatedbicarbonate and the aqueous layer extracted with DCM. The organic layerwas dried with Na₂SO₄, evaporated in vacuo to yield a pale yellow foam.The foam was loaded onto a pretreated manual column (ϕ=4.6×17) preparedwith 2% triethylamine/49% EtOAc/hexanes v/v. The impurities were elutedwith 80% EtOAc/hexanes v/v (8 CV) followed by 100% EtOAc (8 CV). Then 8Awas eluted with 2% MeOH/DCM v/v (5 CV), and 4% MeOH/DCM v/v (8 CV). 8Awas evaporated in vacuo to yield 900 mg (0.58 mmol, 78%) of the amiditeas a diastereomeric mixture.

¹H NMR (500 MHz, DMSO-d₆) δ 8.88 (d, J=1.9 Hz, 1H), 8.41 (s, 1H), 8.32(d, J=9.1 Hz, 1H), 8.01 (d, J=9.3 Hz, 1H), 7.91 (t, J=8.4 Hz, 4H),7.75-7.52 (m, 8H), 7.47 (t, J=7.7 Hz, 2H), 7.36 (dt, J=12.4, 7.4 Hz,4H), 7.21 (1, J=8.6 Hz, 7H), 6.81 (q, J=7.7, 7.1 Hz, 4H), 6.06 (dd,J=8.8, 5.3 Hz, 1H), 5.75 (d, J=3.3 Hz, 1H), 5.37 (dd, J=11.1, 3.3 Hz,1H), 4.84 (q, J=4.7 Hz, 1H), 4.74 (d, J=8.5 Hz, 1H), 4.63 (dd, =10.1,5.1 Hz, 1H), 4.49-4.41 (m, 2H), 4.38-4.15 (m, 3H), 3.80 (dd, J=17.4, 8.2Hz, 3H), 3.70 (d, J=2.9 Hz, 7H), 3.67-3.19 (m, 14H), 3.17 (s, 3H), 3.11(s, 3H), 2.77 (t, J=6.0 Hz, 1H), 2.60 (t, J=5.9 Hz, 1H), 2.03 (s, 2H),1.90 (s, 1H), 1.69 (s, 3H), 1.49 (s, 4H), 1.43-1.34 (m, 2H), 1.23 (dt,J=14.6, 6.6 Hz, 3H), 1.01 (d, J=6.7 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆)δ 172.00, 171.70, 169.39, 165.20, 165.16, 164.87, 159.29, 158.05,158.03, 157.93, 151.89, 151.83, 151.13, 151.05, 144.73, 144.69, 141.97,141.72, 135.42, 135.34, 133.75, 133.49, 133.45, 129.70, 129.66, 129.62,129.20, 129.16, 129.02, 128.95, 128.80, 128.68, 128.57, 127.69, 127.62,126.63, 125.93, 125.85, 118.86, 118.67, 113.05, 100.89, 86.40, 86.13,85.62, 85.59, 82.88, 82.65, 78.92, 78.75, 71.84, 71.01, 70.54, 70.37,70.06, 69.98, 69.88, 68.73, 67.91, 63.00, 62.83, 62.03, 58.83, 58.65,58.22, 58.03, 54.96, 54.86, 49.74, 46.12, 45.65, 42.74, 42.62, 42.58,42.46, 40.62, 38.30, 34.99, 34.52, 29.06, 28.97, 28.57, 26.18, 25.09,25.06, 24.37, 24.29, 24.23, 24.16, 22.67, 21.83, 21.05, 19.89, 19.82,19.78, 19.71, 19.07, 10.57. ³¹P NMR (160 MHz, DMSO-d₆) δ 154.09, 153.88.LRMS calculated for C₈₃H₉₉N₁₀O₁₇P 1538.6927, found m/z 1539.3 (M),1562.3 (M+23)^(Na+), 1573.3 (M+35)^(Cl−). R_(f)=0.35 in 5% MeOH/DCM v/v.

Example 68

(3B)—A mixture of 2A and 2B (12 g, 24.2 mmol) was purified again in thesame manner previously described to remove as much of the T-O-alkylatedisomer as possible. A mixture containing mostly 2B (2.8 g, 5.7 mmol) wastritylated in the same manner as compound 2A to yield 2.6 g of 3B (3.3mmol, 58%).

¹H NMR (400 MHz, DMSO-d₆) δ 8.26 (s, 1H), 8.09 (s, 1H), 7.82 (d, J=7.5Hz, 4H), 7.34-7.15 (m, 11H), 6.80 (d, J=6.8 Hz, 4H), 5.89 (d, J=4.1 Hz,1H), 5.46 (d, J=5.8 Hz, 1H), 4.84 (d, J=4.6 Hz, 1H), 4.14 (t, J=4.7 Hz,1H), 4.09-4.03 (m, 1H), 3.69 (s, 6H), 3.61-3.51 (m, 3H), 3.15 (dd,J=10.1, 4.3 Hz, 1H), 1.58-1.45 (m, 4H), 1.25 (d, J=8.0 Hz, 41-1). ¹³CNMR (100 MHz, DMSO-d₆) δ 167.92, 158.01, 156.06, 152.59, 149.25, 144.78,139.57, 135.47, 134.33, 131.58, 129.63, 127.73, 127.62, 126.61, 122.95,119.16, 113.08, 88.22, 85.48, 80.83, 77.64, 71.70, 69.58, 63.09, 54.96,52.00, 37.32, 29.05, 27.88, 26.07, 25.06, 7.15. LRMS calculated forC₄₅H₄₆N₆O₈ 798.3377, found m/z 799.2 (M+1)⁺, 821.1 (M+23)^(Na+).R_(f)=0.30 in 5% MeOH/DCM v/v

(4B)—Compound 3B was deprotected in a similar fashion as compound 3A toyield crude 4B*. Crude 4B* was then coupled to the GalNAc derivative asdescribed previously in 3A* to yield 2.5 g of 4B (1.9 mmol, 80%).

NMR (400 MHz, DMSO-d₆) δ 8.27 (s, 110, 8.10 (s, 1H), 8.03-7.88 (m, 5H),7.75-7.44 (m, 10H), 7.42-7.16 (m, 13H), 6.82 (dd, J=8.9, 2.4 Hz, 4H),5.90 (d, J=4.4 Hz, 1H), 5.75 (d, J=3.2 Hz, 1H), 5.47 (d, J=6.0 Hz, 1H),5.37 (dd, J=11.1, 3.2 Hz, 1H), 4.87 (q, J=5.0 Hz, 1H), 4.73 (d, J=8.5Hz, 1H), 4.50-4.41 (m, 2H), 4.39-4.22 Om 2H), 4.15 (t, J=5.0 Hz, 1H),4.07 (q, J=4.4 Hz, 1H), 3.84-3.75 (m, 1H), 3.71 (s, 6H), 3.64-3.56 (m,1H), 3.51 (d. J=9.7 Hz, 1H), 3.45-3.38 (m, 1H), 3.27 (dd, J=10.4, 3.6Hz, 1H), 3.17 (dd, J=10.4, 4.7 Hz, 1H), 3.00 (q, J=6.5 Hz, 2H), 2.05 (s,2H), 1.70 (s, 3H), 1.51 (s, 6H), 1.40-1.18 (m, 6H). ¹³C NMR (125 MHz,DMSO-d₆) δ 171.74, 169.41, 165.21, 165.16, 164.87, 158.01, 156.05,152.58, 152.57, 149.25, 144.76, 139.58, 139.56, 135.48, 135.46, 133.78,133.52, 133.48, 129.62, 129.19, 129.17, 129.04, 129.00, 128.99, 128.97,128.70, 128.60, 127.75, 127.62, 126.63, 119.15, 113.09, 100.90, 88.16,85.49, 80.86, 77.70, 71.85, 71.71, 69.96, 69.66, 68.77, 67.91, 63.11,62.02, 54.97, 49.73, 35.03, 29.24, 29.17, 28.59, 26.30, 25.25, 22.70,21.87. LRMS calculated for C₃₇H₄₄N₆O₆ 668.3322 found m/z 691.2 (M+1)⁺,703.2 (M+35)^(Cl−) (4B*). LRMS calculated for C₇₁H₇₇N₇O₁₆ 1283.5427,found m/z 1306.2 (M+23)^(Na+). 1282.2 (M−1)⁻, 1318.2 (M+35)^(Cl−).R_(f)=0.20 in 5% MeOH/DCM v/v

(5B)—Compound 4B (2.5 g, 1.9 mmol) was protected in the same way ascompound 4A to yield 2.0 g of SB (1.5 mmol, 77%).

¹H NMR (400 MHz, DMSO-d₆) δ 8.89 (s, 1H), 8.37 (d, J=5.5 Hz, 2H),8.03-7.87 (m, 5H), 7.73-7.45 (m, 10H), 7.40-7.29 (m, 4H), 7.21 (dd,J=15.7, 8.2 Hz, 7H), 6.81 (dd, J=8.8, 4.4 Hz, 4H), 5.95 (d, J=4.5 Hz,1H), 5.75 (d, J=3.2 Hz, 1H), 5.50 (d, J=5.9 Hz, 1H), 5.36 (dd, J=11.1,3.2 Hz, 1H), 4.90 (q, J=5.1 Hz, 1H), 4.73 (d, J=8.5 Hz, 1H), 4.45 (q,J=8.1, 7.2 Hz, 2H), 4.39-4.23 (m, 2H), 4.15 (t, J=4.9 Hz, 1H), 4.08 (q,J=4.4 Hz, 1H), 3.84-3.75 (m, 1H), 3.70 (s, 6H), 3.65-3.56 (m, 1H),3.54-3.47 (m, 1H), 3.17 (s, 4H), 3.11 (s, 3H), 3.00 (q, J=6.5 Hz, 2H),2.05 (s, 2H), 1.70 (s, 3F1), 1.57-1.43 (m, 6H), 1.40-1.19 (m, 6H). ¹³CNMR (100 MHz, DMSO-d₆) δ 171.77, 169.42, 165.21, 165.17, 164.88, 159.21,158.02, 158.00, 157.92, 151.90, 151.27, 144.74, 141.47, 135.51, 135.46,133.78, 133.52, 133.48, 129.63, 129.58, 129.19, 129.17, 129.04, 129.00,128.97, 128.70, 128.60, 127.75, 127.63, 126.65, 125.76, 113.09, 100.90,88.23, 85.49, 80.95, 77.75, 71.86, 71.65, 69.98, 69.69, 68.77, 67.92,63.15, 62.04, 54.97, 49.75, 45.68, 40.64, 35.04, 34.54, 29.23, 29.16,28.60, 26.30, 25.24, 22.70, 21.88, 11.35. LRMS calculated forC₇₄H₂₈₂N₈O₁₆ 1338.5849. found m/z 1341.3 (M+1)⁺, 1361.3 (M+23)^(Na+),1337.4 (M−1)⁻, 1373.4 (M+35)^(Cl−). R_(f)=0.40 in 7% MeOH/DCM v/v.

(6B)—Compound 5B (500 mg, 0.37 mmol) was treated with succinic anhydridein the same manner as compound 5A to yield 540 mg of 6B (0.35 mmol, 94%)

¹H NMR (400 MHz, DMSO-d₆) δ 8.90 (s, 1H), 8.42 (d, J=9.1 Hz, 2H), 8.06(d, J=9.3 Hz, 1H), 7.91 (t, J=6.9 Hz, 4H), 7.77-7.45 (m, 10H), 7.37 (t,J=7.7 Hz, 2F1), 7.30-7.10 (m, 9H), 6.85-6.73 (m, 4H), 6.19 (d, J=3.0 Hz,1H), 6.12-6.04 (m, 1H), 5.75 (d, J=2.2 Hz, 1H), 5.36 (dd, J=11.1, 3.1Hz, 1H), 4.75 (d, J=8.5 Hz, 1H), 4.70-4.63 (m, 1H), 4.49-4.40 (m, 2H),4.40-4.23 (m, 3H), 4.10-4.03 (m, 1H), 3.83-3.76 (m, 2H), 3.69 (s, 6H),3.54-3.48 (m, 2H), 3.18 (s, 3H), 3.11 (s, 4H), 2.98 (q, J=6.4, 5.5 Hz,3H), 2.80 (q, J=7.2 Hz, 4H), 2.57 (d, J=3.5 Hz, 2H), 2.05 (s, 2H), 1.69(s, 3H), 1.50 (s, 4H), 1.41 (s, 2H), 1.33 (s, 2H), 1.26-1.14 (m, 7H),¹³C NMR (125 MHz, DMSO-d₆) δ 174.17, 172.71, 172.37, 170.34, 166.14,166.09, 165.80, 160.24, 158.93, 158.91, 158.88, 152.99, 151.83, 145.56,142.79, 136.40, 136.26, 134.71, 134.45, 134.41, 130.51, 130.46, 130.10,129.97, 129.95, 129.93, 129.90, 129.64, 129.53, 128.65, 128.52, 127.56,126.65, 113.99, 101.84, 87.28, 86.31, 81.71, 76.73, 73.86, 72.82, 71.23,70.90, 69.68, 68.84, 63.47, 62.97, 55.89, 52.92, 50.66, 46.35, 35.93,35.49, 30.06, 29.74, 29.67, 29.50, 27.18, 26.12, 23.62, 22.78, 10.55,8.11. LRMS calculated for C₇₈H₈₆N₈O₁₉ 1438.6009. R_(f)=0.38 in 5%MeOH/5% Et₃N/DCM v/v

(7B)—Compound 6B (510 mg, 0.33 mmol) was loaded on to LCAA-CPG in thesame manner as 6A to yield 4.1 g of CPG (71 μmollg).

(8B)—Compound 58 (1.4 g, 1.07 mmol) was phosphitylated in the samemanner as compound 5A to yield 1.2 g of the amidite 8B (0.78 mmol, 73%)as a diastereomeric mixture.

¹H NMR (400 MHz, DMSO-d₆) δ 8.88 (d, J=5.5 Hz, 1H), 8.41-8.30 (m, 2H),7.98-7.89 (m, 5H), 7.72-7.45 (m, 11H), 7.41-7.29 (m, 4H), 7.29-7.14 (m,8H), 6.84-6.78 (m, 4H), 6.09 (dd, J=28.1, 4.6 Hz, 1H), 5.74 (d, J=3.4Hz, 1H), 5.35 (dd, J=11.1, 3.3 Hz, 1H), 5.27-5.14 (m, 1H), 4.72 (d,J=8.5 Hz, 1H), 4.44 (q, J=7.3, 6.8 Hz, 2H), 4.37-4.22 (m, 3H), 4.14-4.09(m, 1H), 3.81-3.72 (m, 2H), 3.70 (s, 6H), 3.56-3.35 (m, 6H), 3.18 (s,3H), 3.12-3.10 (m, 3H), 3.03-2.96 (m, 3H), 2.04 (s, 2H), 1.69 (s, 3H),1.50 (s, 6H), 1.38-1.19 (n, 7H), 1.04 (q, J=7.0 Hz, 9H), 0.75 (d, J=6.7Hz, 3H), ¹³C NMR (125 MHz, DMSO-d₆) δ 171.71, 169.36, 165.17, 165.13,164.84, 159.18, 158.01, 157.86, 151.80, 151.13, 144.68, 144.64, 141.62,135.41, 135.38, 133.73, 133.45, 133.42, 129.57, 129.14, 129.00, 128.92,128.65, 128.55, 127.72, 127.58, 126.63, 125.82, 125.78, 118.75, 118.56,113.06, 100.88, 87.48, 87.12, 85.55, 85.47, 81.28, 80.95, 77.36, 77.02,73.45, 73.34, 72.81, 72.67, 71.83, 69.96, 69.74, 68.73, 67.90, 62.74,62.57, 62.01, 58.73, 58.59, 58.15, 58.00, 54.94, 54.85, 49.74, 42.75,42.66, 42.50, 42.40, 35.02, 34.52, 29.24, 29.14, 28.57, 26.31, 25.28,24.26, 24.20, 24.14, 23.78, 23.73, 22.66, 21.85, 19.77, 19.72, 19.55,19.49. ³¹P NMR (162 MHz, DMSO-d₆) δ 154.92, 154.69. LRMS calculated forC-₈₃H₉₉N₁₀O₁₇P 1538.6927, found m/z 1539.3 (M+1)⁺, 1562.3 (M+23)^(Na+).1573.3 (M+35)^(Cl−). R_(f)=0.25 in 5% MeOH/DCM v/v

Example 69

LRMS calculated for C₂₄H₂₅N₆O₆ 496.52, found m/z 497.2 (M+1)⁺, 519.2(M+23)^(Na+), 486.2 (M−1)⁻, 522.2 (M+35)^(Cl−).

(2A/2B)—Compound 1 (2.0 g, 4.2 mmol) was microwaved (100° C., 200 W) inthe presence of N-(6-Bromohexyl)Phthalimide (2.6 g, 8.4 mmol) in DMF (10ml) for 6 hours. NaI (125 mg, 0.84 mmol) was used to speed up thereaction. DMF was evaporated in vacuo and the crude gum was adsorbed tosilica and purified with a 40 g gold column. 2A. as well as the3′-O-alkylated isomer 2B, eluted together with 5% MeOH in DCM v/v. 670mg (1.4 mmol, 33%) of the regioisomeric mixture was collected in aroughly 1 to 1 ratio by ¹H NMR.

¹H NMR (500 MHz, DMSO-d₆) δ 11.31 (s, 2H), 7.93 (d, J=8.1 Hz, 1H),7.88-7.81 (m, 7H), 5.82 (d, J=5.0 Hz, 1H), 5.73 (d, J=5.3 Hz, 1H), 5.63(dd, J=8.1, 1.9 Hz, 2H), 5.30 (d, J=6.1 Hz, 1H), 5.12 (t, J=4.9 Hz, 2H),5.03 (d, J=5.9 Hz, 1H), 4.15 (q, J=5.4 Hz, 1H), 4.07 (q, J=5.0 Hz, 1H),3.90 (d, J=3.9 Hz, 1H), 3.85 (t, J=4.8 Hz, 2H), 3.75 (t, J=4.6 Hz, 1H),3.68-3.49 (m, 9H), 3.43 (dd, J=17.9, 9.5 Hz, 2H), 1.65-1.43 (m, 7H),1.39-1.21 (m, 7H). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.71, 170.69, 165.87,165.82, 153.48, 153.27, 143.32, 143.14, 137.12, 134.36, 125.75, 104.53,104.44, 90.81, 88.93, 87.82, 85.53, 83.85, 80.17, 75.40, 72.43, 72.33,71.09, 63.50, 63.22, 57.68, 48.54, 40.13, 40.09, 31.95, 31.72, 30.68,28.89, 28.81, 27.90, 27.71. LRMS calculated for C₂₃H₂₇N₃O₈473.1798,found m/z 474.1 (M+1)⁺, 472.0 (M−1)⁻, 508.0 (M+35)^(Cl−). R_(f)=0.40 in5% MeOH/DCM v/v

(3A/3B)—A mixture of compounds 2A and 2B (650 mg, 1.4 mmol) wasinitially coevaporated with pyridine to remove any trace water. Then thenucleoside mixture was cooled to 0° C. in pyridine (14 ml) before theaddition of DMTrCl (510 mg, 1.5 mmol) and allowed to warm to roomtemperature over night. An additional 0.2 eq of DMTrCl was added and thereaction mixture stirred for 4 hours before another 0.2 eq of DMTrCl wasadded. The reaction was then stirred for 2 hours, quenched with MeOH andevaporated in vacuo. The crude mixture was washed with saturated NaHCO₃,extracted with DCM, and the organic layer dried with Na₂SO₄. The mixturewas loaded onto a column (40 g gold) and eluted with 60-70%EtOAc/hexanes v/v to yield 310 mg (0.34 mmol, 29%) of 3A and 340 mg(0.44 mmol, 32%) of 3B.

3A)

¹H NMR (400 MHz, DMSO-d₆) δ 11.35 (s, 1H), 7.89-7.76 (m, 4H), 7.71 (d,J=8.1 Hz, 1H), 7.40-7.28 (m, 4H), 7.24 (dd, J=8.8.2.1 Hz, 5H), 6.89 (d,J=8.6 Hz, 4H), 5.77 (d, J=3.6 Hz, 1H), 5.27 (d, J=8.1 Hz, 1H), 5.09 (d,J=6.7 Hz, 1H), 4.15 (q, J=6.1 Hz, 1H), 3.97-3.92 (m, 1H), 3.90-3.85 (m,1H), 3.73 (s, 6H), 3.61-3.49 (m, 4H), 3.30-3.17 (m, 2H), 1.60-1.46 (m,4H), 1.34-1.22 (m, 4H), ¹³C NMR (125 MHz, DMSO-d₆) δ 167.87, 162.92,158.10, 158.09, 150.22, 144.60, 140.15, 135.31, 135.04, 134.28, 131.55,129.73, 127.85, 127.66, 126.75, 122.92, 113.22, 113.20, 101.43, 101.40,87.11, 87.09, 85.84, 82.56, 80.79, 69.71, 68.43, 62.58, 55.02, 55.00,28.88, 27.86, 26.01, 24.92. LRMS calculated for C₄₄H₄₅N₃O₁₀ 775.3105,found m/z 798.0 (M+23)^(Na+), 774.2 (M−1)⁻, 810.1 (M+35)^(Cl−) (3A).R_(f)=0.28 in 60% EtOAc/hexanes v/v (3A)

3B)

¹H NMR (400 MHz, DMSO-d₆) δ 11.34 (s, 1H), 7.88-7.71 (m, 5H), 7.38-7.18(m, 9H), 6.87 (dd, J=8.8, 1.5 Hz, 4H), 5.68 (d, J=3.5 Hz, 114), 5.40 (d,J=5.7 Hz, 1H), 5.29 (d, J=8.1 Hz, 1H), 4.22 (q, J=5.1 Hz, 1H), 3.97 (d,J=6.5 Hz, 1H), 3.92-3.86 (m, 1H), 3.71 (s, 6H), 3.54 (q, J=8.2, 7.2 Hz,3H), 3.34 (t, J=6.7 Hz, 1H), 3.30-3.18 (m, 2H), 1.58-1.40 (m, 4H),1.28-1.19 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.90, 163.02, 158.11,150.41, 144.59, 140.45, 135.26, 135.09, 134.33, 131.57, 129.72, 127.87,127.65, 126.78, 122.95, 113.21, 101.34, 89.41, 85.92, 80.35, 76.70,72.01, 69.56, 63.47, 62.35, 55.01, 37.32, 30.15, 29.03, 27.87, 26.07,25.07, 18.59. LRMS calculated for C₄₄H₄₅N₃O₁₀ 775.3105, found m/z 798.0(M+23)^(Na+), 774.2 (M−1)⁻, 810.0 (M+35)^(Cl−) (3B). R_(f)=0.16 in 60%EtOAc/hexanes v/v (3B).

(4A)—Compound 3A (1.0 g, 1.3 mmol) was dissolved in pyridine (4.5 ml)and imidazole (260 mg, 3.9 mmol) was added. The mixture was stirred for10 minutes at room temperature before the addition of TBSCl (290 mg, 1.9mmol) and the reaction mixture was then stirred at room temperature overnight. The reaction was quenched with MeOH and evaporated in vacuo. Thecrude product was washed with saturated NaHCO₃ and extracted with DCM.The organic layer was dried with Na₂SO₄ before silica gel purification.The product 4A eluted with 35-45% EtOAc/hexanes v/v to yield 1.0 g ofproduct (1.1 mmol, 87%).

¹H NMR (400 MHz, DMSO-d₆) δ 11.35 (s, 1H), 7.89-7.76 (m, 5H), 7.39-7.27(m, 4H), 7.27-7.18 (m, 5H), 6.93-6.83 (m, 4H), 5.74 (s, 1H), 5.27 (d,J=8.1 Hz, 1H), 4.28-4.21 (m, 1H), 3.89 (t, J=6.2 Hz, 2H), 3.72 (d, J=0.9Hz, 6H), 3.62-3.49 (m, 3H), 3.46-3.35 (m, 2H), 3.17 (dd, J=10.9, 3.8 Hz,1H), 1.60-1.42 (m, 4H), 1.26 (dd, J=15.9, 8.0 Hz, 4H), 0.71 (s, 9H),−0.06 (d, J=28.2 Hz, 6H). ¹³C NMR (125 MHz, d_(mso)) δ 173.26, 168.44,163.58, 155.55, 149.75, 145.74, 140.49, 140.30, 139.68, 136.94, 135.19,135.15, 133.22, 133.12, 132.24, 128.32, 118.58, 118.56, 91.39, 87.57,85.97, 75.01, 74.88, 60.43, 60.42, 42.70, 34.50, 33.29, 31.46, 30.82,30.54, 22.96, 0.58, 0.00. LRMS calculated for C₅₀H₅₉N₃O₁₀Si 889.3970,found m/z 912.2 (M+23)^(Na+), 888.3 (M−1)⁻, 925.2 (M+35)^(Cl−).R_(f)=0.72 in 60% EtOAc/hexanes v/v

(5A)—1,2,4-triazole (9.1 g, 131 mmol) was added to acetonitrile (50 ml)and cooled to 0° C. POCl₃ (4.6 g, 30.2 mmol) was added to form ahomogeneous solution. After 20 minutes at 0° C., Et₃N (13.2 g, 131 mmol)was added resulting in the formation of Et₃NHCl salt. In a separateflask, compound 4A (11.7 g, 13.1 mmol) was dissolved in acetonitrile (80ml) and slowly added to the triazole containing flask and allowed tostir for 3 hours. Acetonitrile was removed in vacuo and the resultingcrude foam was dissolved in EtOAc. The salt as filtered off and theorganic layer was washed with saturated NaHCO₃ then dried with Na₂SO₄.EtOAc was removed in vacuo to yield 5A* as a light yellow foam.

LRMS calculated for C₅₂H₆₀N₆O₉Si 940.4191, found in/z 963.0(M+23)^(Na+), 940.2 (M−1)⁻. 975.1 (M+35)^(Cl−). R_(f)=0.50 in 60%EtOAc/hexanes v/v.

5A* was then dissolved in dioxane (132 ml) and NH₄OH (33 ml, 28% w/v)was added. The reaction was allowed to stir over night at roomtemperature. Dioxane was evacuated in vacuo and the crude product waswashed with water and extracted with DCM. Brine was used to clear anyemulsions. The organic layer was dried with Na₂SO₄ and purified (elutedwith 2.5% MeOH/DCM v/v) to yield 8.4 g of 5A (9.4 mmol, 72%). Crude LCMSsuggested some phthalimide damage had occurred (LRMS calculated forC₅₀H₆₃N₅O₉Si 906.15, found m/z 928.1 (M+23)^(Na+), 904.2 (M−1)⁻, 941.0(M+35)^(Cl−)).

NMR (400 MHz, DMSO-d₆) δ 7.91 (d, J=7.4 Hz, 1H), 7.87-7.74 (m, 4H),7.40-7.15 (m, 11H), 6.92-6.83 (m, 4H), 5.80 (d, J=1.6 Hz, 1H), 5.50 (d,J=7.4 Hz, 1H), 4.24 (dd, J=7.6, 5.1 Hz, 1H), 3.91 (d, J=7.4 Hz, 1H),3.72 (s, 8H), 3.53 (t, J=7.0 Hz, 2H), 3.45-3.40 (m, 2H), 3.12 (dd,J=10.9, 3.4 Hz, 1H), 1.62-1.41 (m, 4H), 1.36-1.20 (m, 5H), 0.69 (s, 9H),−0.05 (s, 3H), −0.13 (s, 3H), ¹³C NMR (125 MHz, DMSO-d₆) δ 167.90,165.53, 158.21, 154.70, 144.37, 140.64, 135.15, 135.06, 134.32, 131.57,129.78, 129.74, 127.84, 127.78, 126.88, 122.96, 113.21, 113.18, 85.93,81.59, 81.38, 69.55, 55.07, 55.06, 37.37, 29.23, 27.95, 26.16, 25.43,25.27, 17.59, −4.80, −5.38. LRMS calculated for C₅₀H₆₀N₄O₉Si 888.4130,found m/z 889.0 (M+1)⁺, 911.0 (M+23)^(Na+), 888.1 (M−1)⁻, 923.1(M+35)^(Cl−). R_(f)=0.34 in 5% MeOH/DCM v/v

(6A)—Compound 5A (8.4 g, 9.4 mmol) was refluxed in MeOH (90 ml) withhydrazine (1.5 g, 47.1 mmol) for 3 hours. MeOH was removed in vacuo andthe crude compound was washed with NH₄OH and the aqueous layer wasextracted with DCM. The organic layer was dried with Na₂SO₄ andevaporated in vacuo to yield the crude product 6A*

LRMS calculated for C₄₂H₅₈N₄O₇Si 758.4075, found m/z 759.2 (M+1)⁺, 757.2(M−1)⁻, 793.2 (M+35)^(Cl−). R_(f)=0.11 in 15% MeOH/DCM v/v

6A* was dissolved in DCM (90 ml) with Et₃N (3.9 ml, 28.3 mmol) andstirred under argon briefly. GalNAc(OBz)-C5-NHS ester (7.6 g, 10.4 mmol)was then added and the reaction was allowed to proceed over night. Anadditional 0.2 eq of GalNAc(OBz)-C5-NHS ester was added to drive thereaction closer to completion but the TLC profile remained the same. Theorganic layer was washed with saturated NaHCO₃ and dried with Na₂SO₄.The crude product was purified with silica gel chromatography (elatedwith 2.5% MeOH/DCM v/v) to yield 8.4 g of 6A (6.1 mmol, 65%)

¹H NMR (400 MHz, DMSO-d₆) δ 8.02-7.88 (m, 6H), 7.74-7.16 (m, 22H),6.95-6.81 (m, 4H), 5.82 (s, 1H), 5.74 (d, J=2.6 Hz, 1H), 5.51 (d, J=7.4Hz, 1H), 5.36 (dd, J=11.1, 3.3 Hz, 1H), 4.73 (d, J=8.5 Hz, 1H), 4.44 (q,J=8.6.7.5 Hz, 2H), 4.39-4.21 (m, 3H), 3.92 ((d, J=7.5 Hz, 1H), 3.78 (d,J=9.8 Hz, 1H), 3.72 (s, 8H), 3.46 (dd, J=30.5, 9.4 Hz, 3H), 3.17-2.93(m, 4H), 2.04 (s, 2H), 1.69 (s, 3H), 1.49 (s, 6H), 1.40-1.20 (m, 6H),0.71 (s, 9H). −0.07 (d, J=37.7 Hz, 6H). ¹¹C NMR (125 MHz, DMSO-d₆) δ171.53, 169.19, 165.27, 165.02, 164.97, 164.68, 158.01, 154.45, 144.18,140.48, 134.95, 134.86, 133.58, 133.31, 133.28, 129.58, 129.55, 129.03,129.00, 128.98, 128.85, 128.83, 128.78, 128.51, 128.40, 127.65, 127.59,126.68, 113.02, 112.99, 100.71, 93.65, 93.62, 88.11, 88.09, 85.74,81.43, 81.23, 71.68, 69.80, 69.46, 69.12, 68.55, 67.75, 61.86, 61.35,54.87, 54.86, 49.57, 45.40, 34.83, 29.16, 28.99, 28.39, 26.13, 25.27,25.22, 22.52, 22.51, 21.67, 17.43, 8.36, −4.95, −5.55. LRMS calculatedfor C₇₆H₉₁N₅O₁₇Si 1373.6179. found m/z 1396.2 (M+23)^(Na+), 1372.3(M−1)⁻, 1408.3 (M+35)^(Cl−). R_(f)=0.29 in 5% MeOH/DCM v/v

(7A)—Compound 6A (500 mg, 0.36 mmol) was dissolved in THF (8 ml) andEt₃N.3HF was added to the reaction mixture. Silyl cleavage took 48 hoursto complete. THF was evaporated in vacuo and the crude product waswashed with water and extracted with DCM. The organic layer was driedwith Na₂SO₄ and purified (elute with 5% MeOH/DCM v/v) to yield 440 mg of7A (0.35 mmol, 96%).

¹H NMR (400 MHz, DMSO-d₆) δ 8.02-7.88 (m, 5H), 7.78 (d, J=7.5 Hz, 1H),7.74-7.54 (m, 8H), 7.49 (t, J=7.7 Hz, 2H), 7.43-7.36 (m, 4H), 7.32 (t,J=7.6 Hz, 2H), 7.26 (dd, J 8.9, 2.3 Hz, 5H), 7.17 (d, J=20.1 Hz, 2H),6.90 (d, J=8.7 Hz, 4H), 5.81 (d, J=2.4 Hz, 1H), 5.75 (d, J=4.1 Hz, 1H),5.49 (d, J=7.4 Hz, 1H), 5.37 (dd, J=11.1, 3.3 Hz, 1H) 5.00 (d, J=7.0 Hz,1H), 4.73 (d, J=8.5 Hz, 1H), 4.46 (d, J=7.6 Hz, 2H), 4.38-4.22 (m, 2H),4.16 (q, J=7.1 Hz, 1H), 3.99-3.91 (m, 1H), 3.74 (s, 8H), 3.68-3.47 (m,3H), 3.26 (d, J=2.8 Hz, 2H), 3.01 (q, J=6.6 Hz, 2H), 2.05 (s, 2H), 1.70(s, 3H), 1.51 (s, 6H), 1.32 (dt, J=42.4, 8.9 Hz, 6H), ¹³C NMR (100 MHz,DMSO-d₆) δ 171.74, 169.40, 165.41, 165.19, 165.15, 164.85, 158.09,154.72, 144.62, 140.55, 135.39, 135.16, 133.76, 133.48, 129.70, 129.18,129.15, 129.02, 129.00, 128.98, 128.96, 128.69, 128.58, 127.86, 127.68,126.77, 113.20, 100.87, 93.72, 87.86, 85.78, 81.73, 81.63, 71.85, 69.96,69.73, 68.73, 68.25, 67.90, 62.25, 62.02, 55.01, 54.88, 49.73, 45.59,35.01, 29.13, 28.56, 26.25, 25.17, 22.68, 21.84, 8.54. LRMS calculatedfor C₇₀H₇₇N₅O₁₇ 1259.5314. found m/z 1282.1 (M+23)^(Na+), 1258.2 (M−1)⁻.1294.2 (M+35)^(Cl−). R_(f)=0.33 in 7% MeOH/DCM v/v

(8A)—Compound 7A (6.4 g, 5.1 mmol) was dissolved in DMF (50 ml) andN,N-Dimethylformamide dimethyl acetal (3.0 g, 25.5 mmol) was added. Thereaction mixture was stirred at 50° C. for 2 hours. The crude productwas purified with a silica column (eluted with 2-4% MeOH/DCM v/v) toyield 5.8 g of 8A (4.4 mmol, 86%).

¹H NMR (400 MHz, DMSO-d₆) δ 8.60 (s, 1H), 8.06-7.88 (m, 6H), 7.75-7.45(m, 10H), 7.42-7.21 (m, 11H), 6.90 (d, J=8.8 Hz, 4H), 5.84 (d, J=2.3 Hz,1H), 5.75 (s, 1H), 5.61 (d, J=7.2 Hz, 1H), 5.36 (dd, J=11.1, 3.3 Hz,1H), 5.06 (d, J=6.9 Hz, 1H), 4.73 (d, J=8.5 Hz, 1H), 4.45 (q, J=8.9, 7.6Hz, 2H), 4.40-4.13 (m, 3H), 4.03-3.96 (m, 1H), 3.74 (s, 8H), 3.69-3.56(m, 2H), 3.51 (d, 1=9.8 Hz, 1H), 3.29-3.24 (m, 1H), 3.16 (s, 3H), 3.01(d, J=9.8 Hz, 5H), 2.05 (s, 2H), 1.70 (s, 3H), 1.50 (d, J=5.5 Hz, 6H),1.39-1.22 (m, 6H). ¹³C NMR (125 MHz, DMSO-d₆) δ 171.76, 171.07, 169.40,165.22, 165.17, 164.87, 158.13, 157.87, 154.87, 144.65, 141.75, 135.43,135.10, 133.77, 133.50, 133.48, 129.80, 129.76, 129.21, 129.17, 129.04,129.02, 128.97, 128.70, 128.59, 127.89, 127.71, 126.79, 113.26, 113.24,101.40, 100.90, 88.33, 85.85, 81.97, 81.72, 71.88, 70.00, 69.82, 68.75,68.31, 67.94, 62.24, 62.05, 55.05, 52.03, 49.76, 45.47, 35.03, 34.78,29.14, 29.11, 28.59, 26.25, 25.18, 22.70, 21.86, 8.54, 7.18. LRMScalculated for C₇₃H₈₂N₅O₁₇ 1314.5736, found m/z 1315.2 (M+1)⁺1337.1(M+23)^(Na+), 1314.2 (M−1)⁻, 1349.2 (M+35)^(Cl−). R_(f)=0.41 in 7%MeOH/DCM v/v.

(9A)—Compound 8A (1.0 g, 0.76 mmol) was dissolved in DCM (7.5 ml) and4-(Dimethylamino)pyridine (280 mg, 2.3 mmol) was added. The reactionmixture was stirred for 20 minutes and then succinic anhydride (150 mg,1.5 mmol) was added. The reaction mixture was stirred at roomtemperature for 3 hours. DCM was evaporated in vacuo and the crude foamwas loaded onto a 2% triethylamine in DCM v/v pretreated manual column(ϕ=4.6×20). A gradient of 1-5% MeOH/2-5% triethylamine/DCM v/v was usedto purify 9A. 9A came between 3-4% MeOH/3-4% triethylamine/DCM v/v. 1.1g of 9A (0.71 mmol, 94%) was collected.

¹H NMR (400 MHz, DMSO-d₆) δ 8.63 (s, 1H), 8.05 (d, J=9.3 Hz, 1H), 7.92(q, J=6.3, 5.7 Hz, 5H), 7.75-7.47 (m, 10H), 7.41-7.21 (m, 11H), 6.90 (d,J=8.9 Hz, 4H), 5.90 (d, J=4.2 Hz, 1H), 5.74 (s, 1H), 5.38 (dd, J=11.1,3.3 Hz, 1H), 5.17 (t, J=5.6 Hz, 1H), 4.76 (d, J=8.5 Hz, 1H), 4.46 (q,J=9.5, 7.9 Hz, 2H), 4.38-4.25 (m, 2H), 4.16 (q, J=5.2 Hz, 2H), 3.80 (d,J=9.7 Hz, 1H), 3.74 (s, 6H), 3.52 (dd, J=9.6, 6.1 Hz, 2H), 3.42-3.28 (m,4H), 3.16 (s, 3H), 3.01 (d, J=16.5 Hz, 5H), 2.61-2.52 (m, 2H), 2.06 (s,2H), 1.71 (s, 3H), 1.57-1.15 (m, 14H). ¹³C NMR (100 MHz, DMSO-d₆) δ173.27, 171.74, 171.56, 171.23, 169.39, 165.20, 165.16, 164.87, 158.14,158.05, 154.88, 144.54, 141.83, 135.19, 135.00, 133.77, 133.49, 129.70,129.20, 129.17, 129.03, 129.01, 128.97, 128.70, 128.59, 127.93, 127.60,126.81, 113.29, 101.96, 100.91, 88.17, 86.07, 80.16, 79.25, 71.88,70.28, 70.13, 69.98, 68.73, 67.92, 62.25, 62.04, 55.03, 54.90, 51.99,49.74, 45.46, 34.99, 34.82, 29.12, 28.95, 28.84, 28.71, 28.57, 26.19,25.13, 22.69, 21.84, 10.00, 7.17. LRMS calculated for C₇₇H₈₆N₆O₂₀1414.5897. found m/z 1416.2 (M+1)⁺, 1437.1 (M+23)^(Na+), 1413.3 (M−1)⁻.R_(f)=0.48 in 5% MeOH/5% Et₃N/DCM v/v

(10A)—Compound 9A (1.0 g, 0.66 mmol) was dissolved in acetonitrile (65ml) and HBTU (500 mg, 1.3 mmol) and DIEA (250 mg, 1.9 mmol) were added.The mixture was shaken for 5 minutes then LCAA-CPG (8.2 g, 540 Å, 131μmol/g) was added and shaken over night at room temperature. The CPG wasfiltered and washed with 200 ml each of DCM. 20% MeOH/DCM v/v, anddiethyl ether then dried in vacuo. The CPG was shaken for 1 hour inacetic anhydride (25 ml), pyridine (75 ml), and triethylamine (1 ml)before being washed again by the same conditions as before. Compound 10Awas dried in vacuo overnight and loading was measured with aspectrophotometer (84 μmol/g).

(11A)—Compound 8A (4.0 g, 3.04 mmol) was coevaporated with acetonitriletwice then dissolved in anhydrous DCM (30 ml) under a strict argonatmosphere and cooled to 0° C. 2-CyanoethylN,N,N′,N′-tetraisopropylphosphordiamidite (1.8 g, 6.08 mmol) was addedand the reaction mixture was stirred for 20 minutes then4,5-Dicyanoimidazole (360 mg, 3.04 mmol) was added to the reaction. Thereaction was slowly warmed to room temperature and stirred over night.The reaction mixture was washed with saturated NaHCO₃ and the organiclayer was dried over Na₂SO₄ before being evaporated in vacuo. Theresultant foam was purified with a manual column (ϕ=4.6×30) preparedwith 2% triethylamine/49% EtOAc/hexanes v/v. The impurities were elutedwith 50% EtOAc/hexanes v/v (1 CV), 80% EtOAc/hexanes v/v (7 CV) followedby 100% EtOAc (8 CV). Then 11A was eluted with 2% MeOH/DCM v/v (5 CV),and 4% MeOH/DCM v/v (7 CV). 11A was evaporated in vacuo to yield 2.1 mg(1.37 mmol, 45%) of the amidite as a diastereomeric mixture. 7.5%MeOH/DCM v/v eluted another DMTr containing compound that wascharacterized by mass as oxidized 11A (LCMS found m/z 1532.2 (M+1)⁺.1555.2 (M+23)^(Na+)).

¹H NMR (400 MHz, DMSO-d₆) δ 8.61 (s, 1H), 8.07-7.87 (m, 6H), 7.76-7.19(m, 21H), 6.97-6.82 (m, 4H), 5.88 (dd, J=8.4, 2.6 Hz, 1H), 5.75 (d,J=3.1 Hz, 1H), 5.59 (dd, J=10.9, 7.2 Hz, 1H), 5.37 (dd, J=11.1, 3.3 Hz,1H), 4.74 (d, J=8.5 Hz, 1H), 4.49-4.23 (m, 5H), 4.17-4.07 (m, 1H),3.98-3.92 (m, 1H), 3.83-3.77 (m, 1H), 3.73 (d, J=2.5 Hz, 6H), 3.71-3.45(m, 7H), 3.35 (d, J=22.0 Hz, 6H), 3.16 (s, 3H), 3.01 (d, J=5.1 Hz, 5H),2.74 (t, J=5.9 Hz, 1H), 2.64-2.57 (m, 1H), 2.05 (s, 2H), 1.70 (s, 3H),1.51 (s, 6H), 1.42-1.19 (m, 7H), 1.17-1.03 (m, 10H), 0.95 (d, J=6.7 Hz,3H), ¹³C NMR (125 MHz, DMSO-d₆) δ 171.69, 171.08, 169.35, 165.17,165.13, 164.84, 158.14, 157.85, 154.83, 154.81, 144.53, 144.45, 141.75,141.54, 135.15, 134.91, 134.87, 133.71, 133.44, 133.41, 129.81, 129.75,129.17, 129.13, 129.00, 128.91, 128.64, 128.53, 127.81, 127.70, 126.80,118.75, 118.61, 113.16, 101.60, 101.51, 100.87, 88.81, 88.32, 85.99,81.35, 80.96, 80.49, 71.83, 69.97, 69.80, 69.48, 69.38, 68.71, 67.90,62.01, 61.87, 61.28, 58.40, 58.25, 58.17, 58.01, 54.99, 49.73, 45.61,42.61, 42.56, 42.51, 42.45, 34.99, 34.73, 29.20, 29.14, 29.10, 28.56,26.28, 25.27, 25.23, 24.30, 24.24, 24.17, 22.66, 21.83, 19.80, 19.75,³¹P NMR (160 MHz, DMSO-d₆) δ 153.88, 153.40. LRMS calculated forC₈₂H₉₉N₈O₁₈P 1514.6815, found m/z 1537.2 (M+23)^(Na+), 1551.2(M+35)^(Cl). R_(f)=0.41 in 7% MeOH/DCM v/v.

Example 70

(4B)—Compound 3B (4.95 g, 6.38 mmol) was protected with TBSCl (1.44 g,9.57 mmol) in the same manner as compound 3A to yield 5.00 g of 4B (5.62mmol, 88%).

¹H NMR (400 MHz, DMSO-d₆) δ 11.37 (s, 1H), 7.87-7.77 (m, 5H), 7.37-7.19(m, 9H), 6.88 (dd, J=8.9, 1.8 Hz, 4H), 5.66 (d, J=3.7 Hz, 1H), 5.28 (d,J=8.1 Hz, 1H), 4.36 (t, J=4.1 Hz, 1H), 3.99 (t, 0.1=2.9 Hz, 1H),3.88-3.82 (m, 1H), 3.71 (s, 6H), 3.57-3.42 (m, 3H), 3.34 (d, J=10.7 Hz,2H), 3.25 (dd, J=11.0, 3.5 Hz, 1H), 1.57-1.37 (m, 4H), 1.29-1.19 (m,4H), 0.81 (s, 9H), 0.03 (d, J=4.4 Hz, 6H). ¹³C NMR (125 MHz, DMSO-d₆) δ167.85, 162.93, 158.14, 150.33, 144.47, 139.87, 135.04, 134.89, 134.30,131.53, 129.73, 127.85, 127.61, 126.81, 122.91, 113.20, 101.32, 88.72,86.03, 80.60, 76.69, 73.71, 69.85, 62.08, 59.70, 54.99, 37.28, 2907,27.83, 26.04, 25.41, 25.09, 17.63, −5.07, −5.26. LRMS calculated forC₅₀H₅₉N₃O₁₀Si 889.3970, found m/z 912.0 (M+23)^(Na+). 888.2 (M−1)⁻.924.2 (M+35)^(Cl−). R_(f)=0.71 in 60% EtOAc/Hexanes v/v

(5B)—Compound 4B (4.94 g, 5.55 mmol) was converted to 5B through theintermediate 5B* in the same manner as compound 4A. 3.45 g of 5B wasobtained (3.88 mmol, 70%) for the two step reaction.

5B*)

LRMS calculated for C₅₂H₆₀N₆O₉Si 940.4191, found m/z 964.3 (M+23)^(Na+).941.3 (M−1)⁻, 976.3 (M+35)^(Cl−) R_(f)=0.51 in 60% EtOAc/Hexanes v/v

5B)

¹H NMR (400 MHz, DMSO-d₆) δ 7.89-7.75 (m, 5H), 7.38-7.21 (m, 9H), 7.09(s, 2H), 6.88 (d, J=7.9 Hz, 4H), 5.71 (d, J=14.2 Hz, 1H), 5.50 (d, J=7.2Hz, 1H), 4.24 (s, 1H), 3.99 (d, J=6.1 Hz, 1H), 3.88-3.83 (m, 1H), 3.72(s, 6H), 3.52 (t, J=6.8 Hz, 2H), 3.41 (dd, J=26.0, 9.6 Hz, 2H), 3.21 (d,J=11.1 Hz, 2H), 1.46 (d, J=55.9 Hz, 4H), 1.20 (s, 4H), 0.82 (s, 9H),0.05 (d, J=18.7 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.87, 165.54,158.14, 154.95, 144.49, 140.38, 135.13, 135.06, 134.33, 131.56, 129.71,127.86, 127.65, 126.82, 122.94, 113.19, 93.63, 89.79, 85.93, 79.86,76.34, 74.03, 69.80, 61.80, 55.01, 54.90, 37.30, 29.10, 27.84, 26.07,25.53, 25.10, 17.71, −4.77, −5.39. LRMS calculated for C₅₀H₆₀N₄O₉Si888.4130, found m/z 911.2 (M+23)^(Na+). 888.3 (M−1)⁻, 923.3(M+35)^(Cl−). R_(f)=0.46 in 7% MeOH/DCM v/v

(6B)—Compound 5B (3.45 g, 3.88 mmol) was treated with hydrazine in thesame manner as 5A to yield intermediate 6B*.

6B*)

LRMS calculated for C₄₂H₅₈N₄O₇Si 758.4075, found in/z 759.2 (M+1)⁺781.2(M+23)^(Na+). 757.2 (M−1)⁻. 793.2 (M+35)^(Cl−). R_(f)=0.20 in 15%MeOH/DCM v/v

Intermediate 6B* was then coupled to GalNAc(OBz)-C5-NHS ester in thesame manner as 6A* to yield 4.64 g of 6B (3.38 mmol, 87%).

6B)

¹H NMR (500 MHz, DMSO-d₆) δ 7.99-7.86 (m, 614), 7.73-7.53 (m, 9H), 7.48(t, J=7.7 Hz, 2H), 7.41-7.34 (m, 4H), 7.31 (1, J=7.6 Hz, 2H), 7.24 (dd,J=8.8, 2.8 Hz, 6H), 6.89 (d. J=8.3 Hz, 414), 5.75 (d, J=3.1 Hz, 114),5.70 (d, J=2.5 Hz, 1H), 5.51 (d, J=7.4 Hz, 1H), 5.37 (dd, J=11.1, 3.1Hz, 1H), 4.74 (d, J=8.5 Hz, 1H), 4.45 (d, J=7.6 Hz, 2H), 4.38-4.24 (m,3H), 4.01 (d, J=6.6 Hz, 1H), 3.90-3.85 (m, 1H), 3.79 (d, J=10.0 Hz, 1H),3.73 (s, 6H), 3.55-3.42 (m, 2H), 3.38 (d, J=9.5 Hz, 1H), 3.30-3.18 (m,2H), 2.98 (q, J=6.5 Hz, 2H), 2.04 (s, 2H), 1.69 (s, 3H), 1.51 (s, 4H),1.44-1.28 (m, 4H), 1.23-1.14 (m, 4H), 0.83 (s, 9H), 0.10-0.02 (m, 6H).¹¹C NMR (125 MHz, DMSO-d₆) δ 172.22, 169.88, 166.00, 165.71, 165.67,165.38, 158.68, 155.43, 145.00, 140.94, 135.71, 135.60, 134.27, 134.01,133.97, 130.23, 129.73, 129.68, 129.55, 129.47, 129.20, 129.09, 128.39,128.19, 127.36, 113.73, 101.42, 94.19, 90.20, 86.48, 80.46, 76.98,74.58, 72.37, 70.51, 70.44, 69.26, 68.45, 67.51, 62.56, 62.43, 55.55,55.54, 50.28, 38.87, 35.54, 29.83, 29.66, 29.11, 26.84, 26.05, 25.82,25.63, 23.21, 22.39, 18.24, −4.26. −4.83. LRMS calculated forC₇₆H₉₁N₅O₁₇Si 1373.6179, found m/z 1396.2 (M+23)^(Na+), 1373.3 (M−1)⁻,1408.3 (M+35)^(Cl−). R_(f)=0.48 in 7% MeOH/DCM v/v

(7B)—Compound 6B (4.60 g, 3.35 mmol) was desilylated with Et₃N.3HF (6.54ml, 40.16 mmol) in THF (67 ml) in a similar manner as 6A to yield 7Bquantitatively.

¹H NMR (500 MHz, DMSO-d₆) δ 8.00 (d, J=9.3 Hz, 1H), 7.91 (t, J=7.4 Hz,4H), 7.82 (d, J=7.4 Hz, 1H), 7.70 (t, J=6.3 Hz, 4H), 7.63 (t, J=7.4 Hz,1H), 7.56 (q, J=8.2 Hz, 3H), 7.48 (t, J=7.7 Hz, 2H), 7.41-7.34 (m, 4H),7.31 (t, J=7.7 Hz, 2H), 7.24 (d, J=7.4 Hz, 6H), 7.10 (s, 1H), 6.88 (d,J=8.0 Hz, 4H), 5.76-5.73 (m, 2H), 5.52 (d, J=7.4 Hz, 1H), 5.39-5.31 (m,2H), 4.74 (d, J=8.5 Hz, 1H), 4.45 (q, J=9.3, 7.9 Hz, 2H), 4.38-4.31 (m,1H), 4.32-4.22 (m, 1H), 4.12 (q, J=5.0 Hz, 1H), 4.01-3.97 (m, 1H),3.93-3.89 (m, 1H), 3.82-3.77 (m, 1H), 3.73 (s, 6H), 3.56-3.48 (m, 2H),3.30 (d, J=9.4 Hz, 2H), 3.19 (dd, J=10.8, 3.5 Hz, 1H), 2.99 (q, J=6.6Hz, 2H), 2.04 (1, J=6.6 Hz, 2H), 1.69 (s, 3H), 1.54-1.39 (m, 6H),1.37-1.29 (m, 2H), 1.21 (d, J=7.1 Hz, 4H). ¹³C NMR (125 MHz, DMSO-d₆) δ171.68, 169.34, 165.53, 165.17, 165.12, 164.83, 158.08, 155.00, 144.52,140.72, 135.34, 135.18, 133.72, 133.46, 133.43, 129.64, 129.61, 129.17,129.13, 128.99, 128.92, 128.65, 128.54, 127.82, 127.64, 126.74, 113.17,100.87, 93.63, 90.33, 85.81, 79.75, 76.44, 72.33, 71.83, 69.95, 69.49,68.71, 67.89, 62.07, 62.01, 54.98, 49.71, 38.33, 34.98, 29.20, 29.11,28.55, 26.26, 25.19, 22.66, 21.82. LRMS calculated for C-₇₀H₇₇N₅O₁₇1259.5314, found m/z 1282.1 (M+23)^(Na+), 1259.2 (M−1)⁻, 1294.1(M+35)^(Cl−). R_(f)=0.24 in 7% MeOH/DCM v/v

(8B)—Compound 7B (4.21 g, 3.34 mmol) was protected at the N4 positionwith N,N-Dimethylformamide dimethyl acetal (1.99 g, 16.70 mmol) in thesame manner as 7A to yield 3.79 g of compound 8A (2.88 mmol, 86%).

¹H NMR (500 MHz, DMSO-d₆) δ 8.62 (s, 1H), 8.02-7.88 (m, 6H), 7.74-7.52(m, 8H), 7.48 (t, J=7.7 Hz, 2H), 7.41-7.21 (m, 11H), 6.93-6.85 (m, 4H),5.75 (d, J=4.6 Hz, 3H), 5.62 (d, J=7.2 Hz, 1H), 5.43 (d, J=5.4 Hz, 1H),5.37 (dd, J=11.1, 3.3 Hz, 1H), 4.73 (d, J=8.5 Hz, 1H), 4.49-4.41 (m,2H), 4.37-4.24 (m, 2H), 4.20-4.15 (m, 1H), 4.07-4.02 (m, 1H), 3.97-3.93(m, 1H), 3.82-3.76 (m, 1H), 3.73 (s, 6H), 3.58-3.48 (m, 2H), 3.31 (s,2H), 3.26 (dd, J=10.8, 3.5 Hz, 1H), 3.15 (s, 3H), 3.05-2.95 (m, 5H),2.09-2.00 (m, 2H), 1.70 (s, 3H), 1.55-1.40 (m, 6H), 1.38-1.29 (m, 2H),1.22 (s, 4H). ¹³C NMR (126 MHz, DMSO-d₆) δ 171.68, 171.04, 169.35,165.17, 165.12, 164.83, 158.09, 157.81, 154.99, 144.53, 141.90, 135.33,135.06, 133.72, 133.46, 133.43, 129.71, 129.66, 129.17, 129.13, 128.99,128.92, 128.65, 128.55, 127.83, 127.63, 126.76, 113.19, 101.28, 100.87,90.81, 85.86, 79.97, 76.32, 72.35, 71.82, 69.95, 69.49, 68.72, 67.90,62.00, 54.99, 54.85, 49.72, 45.67, 35.00, 34.72, 29.20, 29.11, 28.57,26.25, 25.20, 22.66, 21.83. LRMS calculated for C₇₃H₈₂N₆O₁₇ 1314.5736,found m/z 1316.4 (M+1)⁺, 1337.2 (M+23)^(Na+), 1314.2 (M−1)⁻, 1349.2(M+35)^(Cl−). R_(f)=0.32 in 7% MeOH/DCM v/v

(9B)—Compound 8B (500 mg, 0.380 mmol) was succinated under the sameconditions as 9A to yield 550 mg of compound 9B (0.363 mina 95%).

¹H NMR (400 MHz, DMSO-d₆) δ 8.61 (s, 1H), 8.07 (d, J=9.2 Hz, 1H),7.99-7.84 (m, 5H), 7.79-7.44 (m, 10H), 7.41-7.20 (m, 11H), 6.88 (d,J=8.5 Hz, 4H), 5.86 (d, J=2.0 Hz, 1H), 5.75 (d, J=3.4 Hz, 1H), 5.70 (d,J=7.2 Hz, 1H), 5.43-5.33 (m, 2H), 4.75 (d, J=8.5 Hz, 1H), 4.49-4.40 (m,2H), 4.37-4.21 (m, 3H), 4.02-3.96 (m, 1H), 3.72 (s, 7H), 3.59-3.45 (m,2H), 3.43-3.20 (m, 6H), 3.15 (s, 3H), 3.04-2.93 (m, 5H), 2.46-2.38 (m,2H), 2.05 (s, 2H), 1.69 (s, 3H), 1.50 (s, 4H), 1.41-1.27 (m, 4H), 1.16(s, 4H). ¹³C NMR (125 MHz, DMSO-d₆) δ 173.26, 171.72, 171.32, 171.10,169.35, 165.17, 165.13, 164.83, 158.10, 157.92, 154.67, 144.47, 142.44,135.23, 135.05, 133.72, 133.43, 129.69, 129.18, 129.13, 129.00, 128.92,128.65, 128.54, 127.83, 127.65, 126.76, 113.18, 101.67, 100.89, 88.99,85.87, 80.36, 75.39, 73.66, 71.87, 70.48, 69.95, 68.69, 67.90, 62.53,62.02, 61.80, 54.98, 54.86, 51.98, 51.26, 49.72, 40.84, 38.32, 34.95,34.77, 29.10, 29.06, 28.97, 28.88, 28.67, 28.53, 26.21, 25.16, 22.65,21.80, 10.64, 7.14. LRMS calculated for C₇₇H₈₆N₆O₂₀ 14145897, found m/z1438.9 (M+23)^(Na+), 1413.2 (M−1)⁻

(10B)—Compound 9B (500 mg, 0.329 mmol) was loaded on to succinated inthe same fashion as compound 10A to yield 4.11 g of 10B with a loadingof 79 μmol/g.

Example 71

LRMS calculated for C₂₄H₂₈N₆O₆ 496.52. found m/z 497.2 (M+1)⁺, 519.2(M+23)^(Na+), 486.2 (M−1)⁻, 522.2 (M+35)^(Cl−)

(2A/2B)—Compound 1 (40 g, 113.2 mmol) was dissolved in DMF (950 ml). NaH(60% in oil; 11.32 g, 283 mmol) was added and the reaction mixture wasstirred till the evolution of gas ceased, about 3 hours. ThenN-(BromohexylpPhthalimide (52.7 g, 169.8 mmol) was added and thereaction stirred for 3 hours at room temperature. The reaction mixturewas heated to 50° C., and held for 72 hours. DMF was removed in vacuoand the resultant gum was adsorbed to silica for silica gel columnpurification. A mixture of 2A and 2B eluted between 4-6% MeOH/DCM v/v toyield 29.85 g of product (51.2 mmol, 45%),

¹H NMR (400 MHz, DMSO-d₆) δ 12.05 (d, J=9.9 Hz, 1H. D₂O exchangeable),11.65 (d, J=8.8 Hz, 1H, D₂O exchangeable), 8.26 (d, J=9.5 Hz, 1H), 7.82(p, J=4.3 Hz, 4H), 5.87 (d, J=6.5 Hz, 1H), 5.78 (d, J=6.0 Hz, 0H, D₂Oexchangeable), 5.39 (d, J=6.2 Hz, 0H, D₂O exchangeable), 5.12 (d, J=4.6Hz, 1H. D₂O exchangeable), 5.04 (t, J=5.4 Hz, 1H, D₂O exchangeable),4.56-4.51 (m, 0H), 4.33-4.27 (m, 1H), 4.24 (q, J=4.5 Hz, 1H), 3.94-3.87(m, 1H), 3.64-3.42 (m, 6H), 2.74 (p, J=6.8 Hz, 1H), 1.43 (dt, J=27.1,6.5 Hz, 4H), 1.23-1.12 (m, 4H), 1.09 (dd, J=6.6, 4.9 Hz, 6H). ¹³C NMR(125 MHz, DMSO-d₆) δ 180.03, 167.91, 167.89, 167.81, 154.72, 148.83,148.19, 137.43, 137.41, 134.30, 131.55, 131.52, 122.92, 122.86, 119.99,86.24, 84.46, 84.44, 81.41, 69.59, 68.89, 68.88, 61.25, 37.16, 34.70,28.87, 27.84, 27.78, 25.90, 24.79, 18.82, 18.79, 18.75. LRMS calculatedfor C₂₈H₃₄N₆O₈ 582.2438, found m/z 583.2 (M+1)⁺, 581.1 (M−1)⁻, 617.1(M+35)^(Cl−). R_(f)=0.30 in 7% MeOH/DCM v/v

(3A/3B)—Compound 2A/2B (14.1 g, 24.2 mmol) was dissolved in anhydrouspyridine (240 ml) under a strict argon atmosphere and cooled to 0° C.Then DMTrCl (9.0 g, 26.6 mmol) was added and the reaction was warmed toroom temperature and allowed to stir for 72 hours. The reaction wasquenched with MeOH and pyridine removed in vacuo. The crude productswere washed with saturated NaHCO₃ and extracted with DCM. The organiclayer was dried over Na₂SO₄ and evaporated in vacuo. The products werepurified with silica gel chromatography to yield 13.82 g (15.62 mmol,65%) of 3A and 2.06 g (2.33 mmol, 10%) of 3B.

3A)

¹H NMR (400 MHz, DMSO-d₆) δ 12.06 (s, 1H), 11.59 (s, 1H), 8.12 (s, 1H),7.81 (p, J=4.4 Hz, 4H), 7.33 (d, J=7.3 Hz, 2H), 7.27-7.16 (m, 7H), 6.82(dd, J=8.7, 7.1 Hz, 4H), 5.92 (d, J=5.5 Hz, 1H), 5.14 (d, J=5.6 Hz, 1H),4.39 (t, J=5.2 Hz, 1H), 4.27 (q, J=4.8 Hz, 1H), 4.03 (q, J=3.9 Hz, 1H),3.71 (s, 6H), 3.57 (dd, J=11.2, 4.7 Hz, 1H), 3.51-3.38 (m, 3H), 3.27(dd, J=10.4, 6.1 Hz, 1H), 3.15 (dd, J=10.3, 3.0 Hz, 1H), 2.74 (p, J=6.8Hz, 1H), 1.47 (dt, J=21.2, 6.6 Hz, 4H), 1.28-1.15 (m, 4H), 1.10 (dd,J=6.6, 5.1 Hz, 61-1). ¹³C NMR (125 MHz, DMSO-d₆) δ 180.04, 167.83,158.02, 158.00, 154.74, 148.76, 148.16, 144.74, 137.44, 135.44, 135.35,134.30, 131.52, 129.68, 129.65, 127.72, 127.65, 126.62, 122.92, 120.29,113.06, 85.52, 85.02, 84.06, 80.59, 69.78, 69.12, 63.93, 63.45, 37.19,34.72, 28.89, 27.79, 25.94, 24.83, 18.80, 18.78. LRMS calculated forC₄₉H₅₂N₆O₁₀ 884.3745. found m/z 885.2 (M+1)⁺. 907.0 (M+23)^(Na+), 883.2(M−1)⁻. 919.2 (M+35)^(Cl−). R_(f)=0.52 in 100% EtOAc

3B)

¹H NMR (400 MHz, DMSO-d₆) δ 12.08 (s, 1H, D₂O exchangeable), 11.67 (s,1H, D₂O exchangeable), 8.14 (s, 1H), 7.86-7.76 (m, 4H), 7.37-7.14 (m,9H), 6.81 (dd, J=8.8, 4.0 Hz, 4H), 5.81 (d, J=4.7 Hz, 1H), 5.51 (d,J=5.7 Hz, 1H, D₂O exchangeable), 4.67 (d, J=4.7 Hz, 1H), 4.05-3.91 Om2H), 3.70 (s, 6H), 3.55 (dt, J=14.2, 8.2 Hz, 3H), 3.39 (d, J=9.4 Hz,1H), 3.26-3.14 (m, 2H), 2.76 (p, J=6.8 Hz, 1H), 1.50 (dt, J=35.7, 6.6Hz, 4H), 1.32-1.19 (m, 4H), 1.11 (d, J=6.8 Hz, 6H). ¹³C NMR (126 MHz.D₂O) δ 180.10, 167.90, 158.02, 154.81, 148.81, 148.16, 144.70, 137.29,135.39, 135.36, 134.33, 131.55, 129.64, 129.61, 127.74, 127.61, 126.64,122.94, 120.28, 113.07, 87.17, 85.57, 81.14, 77.71, 72.22, 69.63, 63.31,59.72, 54.94, 37.30, 34.73, 29.08, 27.86, 26.05, 25.03, 18.87, 18.78,14.05. LRMS calculated for C₄₄H₅₂N₆O₁₀ 884.3745, found m/z 885.1 (M+1)⁺.883.0 (M−1)⁻. 919.0 (M+35)^(Cl−). R_(f)=0.32 in 100% EtOAc

(4A)—Compound 3A (5.0 g, 5.65 mmol) was treated with hydrazine (900 mg,28 mmol) in refluxing MeOH (56 ml) for 3 hours. Both phthalimide andisohutyryl protecting groups were cleaved during the reaction. MeOH wasremoved in vacuo and the crude product was washed with NH₄OH. Theaqueous layer was extracted with DCM and the organic layer was driedwith Na₂SO₄. DCM was removed in vacuo to yield 4A* as a crude foam.

LRMS calculated for C₃₇H₄₄N₆O₇ 684.3271. found m/z 685.2 (M+1)⁺, 683.1(M−1)⁻, 719.1 (M+35)^(Cl−). R_(f)=0.00 in 7% MeOH/DCM v/v.

4A* was then dissolved in anhydrous DCM (56 ml) under a strict argonatmosphere and Et₃N (2.4 ml, 17 mmol) was added. GalNAc(OBz)-C5-NHSester (4.5 g, 6.22 mmol) was then added and the reaction stirred at roomtemperature over night. DCM was evaporated in vacuo and the crude foamwas washed with saturated NaHCO₃ then the aqueous layer was extractedwith DCM. The organic layer was dried with Na₂SO₄ and then removed invacuo. The crude foam was purified with silica gel chromatography(eluted with 2.5-5% MeOH/DCM v/v) to yield 7.0 g (5.41 mmol, 96%) of 4A.Coevaporation with acetonitrile did not reduce the triethylamine peakseen in ¹H NMR.

¹H NMR (400 MHz, DMSO-d₆) δ 10.73 (s, 1H), 9.90 (s, 1H), 8.02 (d, J=9.3Hz, 1H), 7.91 (t, J=7.0 Hz, 4H), 7.79 (s, 1H), 7.74-7.52 (m, 8H), 7.48(t, J=7.7 Hz, 2H), 7.37 (dd, J=13.9, 7.4 Hz, 4H), 7.30-7.16 (m, 7H),6.84 (dd, J=8.9, 2.7 Hz, 4H), 6.53 (s, 2H), 5.81 (d, J=5.4 Hz, 1H), 5.75(d, J=3.1 Hz, 1H), 5.36 (dd, J=11.1, 3.2 Hz, 1H), 5.13 (d, J=5.7 Hz,1H), 4.74 (d, J=8.5 Hz, 1H), 4.49-4.40 (m, 2H), 4.39-4.19 (m, 4H), 3.99(q, J=4.2 Hz, 1H), 3.78 (d, J=9.6 Hz, 1H), 3.71 (s, 6H), 3.61-3.46 (m,2H), 3.46-3.37 (m, 1H), 3.24-3.12 (m, 2H), 2.97 (q, J=6.6 Hz, 2H), 2.04(s, 2H), 1.69 (s, 3H), 1.56-1.37 (m, 6H), 1.36-1.25 (m, 2H). NMR (125MHz, DMSO-d₆) δ 171.72, 169.38, 165.19, 165.14, 164.84, 158.02, 156.66,153.81, 151.30, 144.77, 135.47, 135.37, 134.89, 133.75, 133.47, 129.67,129.18, 129.16, 129.14, 129.01, 128.98, 128.95, 128.68, 128.57, 127.77,127.65, 126.64, 116.64, 113.12, 100.88, 85.53, 84.44, 83.53, 80.61,71.86, 69.96, 69.78, 69.08, 68.73, 67.90, 63.84, 62.02, 54.98, 49.72,45.42, 38.30, 34.99, 29.12, 29.06, 28.56, 26.17, 25.06, 22.68, 21.82,8.47. LRMS calculated for C₇₁H₇₇N₇O₁₇ 1299.5376. found m/z 1301.1(M+1)⁺1322.1 (M−23)_(Na+). 1298.2 (M−1)⁻, 1336.2 (M+35)^(Cl−).R_(f)=0.37 in 7% MeOH/DCM v/v.

(5A)—Compound 4A (6.8 g, 5.23 mmol) was dissolved in DMF.N,N-Dimethylformamide dimethyl acetal (1.9 g, 15.7 mmol) was added tothe reaction mixture and heated to 60° C. for 1.5 hours. DMF was removedin vacuo and the crude product adsorbed to silica. Purification wascarried out with silica gel chromatography (eluted with 2.5-5% MeOH/DCMv/v) to yield 5.63 g of 5A (4.15 mmol, 79%).

¹H NMR (400 MHz, DMSO-d₆) δ 11.38 (s, 1H), 9.94 (s, 1H), 8.48 (s, 1H),8.03 (d, J=9.3 Hz, 1H), 7.97-7.87 (m, 5H), 7.75-7.52 (m, 8H), 7.48 (t,J=7.7 Hz, 2H), 7.37 (dd, J=14.6, 7.4 Hz, 4H), 7.29-7.16 (m, 7H), 6.83(dd, J=8.7, 4.6 Hz, 4H), 5.93 (d, J=4.9 Hz, 1H), 5.75 (d, J=3.1 Hz, 1H),5.37 (dd, =11.1, 3.2 Hz, 1H), 5.21 (d, J=5.7 Hz, 1H), 4.75 (d, J=8.5 Hz,1H), 4.45 (q, J=8.3, 7.3 Hz, 2H), 4.39-4.23 (m, 4H) 4.02 (q, J=4.7 Hz,1H), 3.78 (d, J=9.5 Hz, 1H), 3.71 (s, 6H), 3.52 (ddt, J=40.7, 16.1, 7.9Hz, 4H), 3.25 (dd, J=10.3, 5.9 Hz, 1H), 3.15 (d, J=7.7 Hz, 1H), 3.07 (s,3H), 3.01 2.92 (m, 5H), 2.04 (s, 2H), 1.70 (s, 3H), 1.55-1.38 (m, 6H),1.35-1.24 (m, 2H). ¹³C NMR (125 MHz, DMSO-d₁) δ 171.74, 169.40, 165.20,165.15, 164.85, 158.03, 158.01, 157.79, 157.58, 157.28, 149.86, 144.76,136.33, 135.47, 135.38, 133.75, 133.48, 133.45, 129.66, 129.62, 129.15,129.14, 129.01, 128.99, 128.94, 128.67, 128.56, 127.76, 127.63, 126.64,119.78, 113.11, 100.88, 85.50, 85.06, 83.46, 80.78, 71.87, 69.97, 69.83,69.04, 68.72, 67.91, 63.80, 62.03, 54.98, 49.73, 45.45, 45.43, 34.99,34.63, 29.10, 29.06, 28.55, 26.16, 25.07, 22.67, 21.82, 8.48. LRMScalculated for C₇₄H₈₂N₈O₁₇ 1354.5798. found m/z 1356.1 (M+1)⁺, 1377.2(M+23)^(Na+). 1353.3 (M−1)⁻, 1389.2 (M+35)^(Cl−). R_(f)=0.40 in 7%MeOH/DCM v/v

(6A)—Compound 5A (1.0 g, 0.74 mmol) was dissolved in DCM (7 ml) and4-Dimethylaminopyridine (270 mg, 2.21 mmol) was added to the reactionmixture and stirred for 5 minutes. Then succinic anhydride (150 mg, 1.48mmol) was added and the reaction mixture was stirred at room temperatureover night. DCM was evaporated in vacuo and the crude foam was loadedonto a 2% triethylamine in DCM v/v pretreated manual column (ϕ=4.6×17).A gradient of 1-5% MeOH/2-5% triethylamine/DCM v/v was used to purify6A. 6A came at 4-5% MeOH/4-5% triethylamine/DCM v/v in quantitativeyield.

¹H NMR (500 MHz, DMSO-d₆) δ 11.43 (s, 1H), 8.62 (s, 1H), 8.08-7.98 (m,2H), 7.92 (t, J=8.0 Hz, 4H), 7.71 (dd, J=12.7, 7.3 Hz, 4H), 7.64 (t,J=7.4 Hz, 1H), 7.57 (q, J=7.7, 7.1 Hz, 3H), 7.50 (d, J=7.7 Hz, 2H), 7.39(t, J=7.8 Hz, 2H), 7.30-7.14 (m, 9H), 6.81 (dd, J=8.8, 4.9 Hz, 4H), 5.94(d, J=4.4 Hz, 1H), 5.76 (s, 1H), 5.63 (t, J=5.5 Hz, 1H), 5.38 (dd,J=11.1, 3.3 Hz, 1H), 4.90 (t, J=4.9 Hz, 1H), 4.76 (d, J=8.5 Hz, 1H),4.46 (q, J=9.9, 8.2 Hz, 2H), 4.39-4.24 (m, 2H), 4.15 (q, =4.9 Hz, 1H),3.82-3.77 (n, 1H), 3.72 (s, 6H), 3.53-3.43 (m, 2H), 3.37 (q, J=7.2 Hz,3H), 3.27 (dd, J=10.5, 5.2 Hz, 1H), 3.22-3.15 (m, 1H), 3.03 (s, 3H),3.01-2.94 (m, 5H), 2.79 (q, J=7.0 Hz, 2H), 2.59 (q, J=6.4, 5.9 Hz, 2H),2.05 (s, 2H), 1.70 (s, 3H), 1.56-1.46 (m, 4H), 1.38 (s, 2H), 1.33-1.26(m, 2H), 1.15 (s, 4H), ¹³C NMR (125 MHz, DMSO-d₆) δ 173.21, 171.83,171.69, 169.34, 165.16, 165.11, 164.81, 158.00, 157.99, 157.96, 157.55,157.24, 149.59, 14458, 137.75, 135.32, 135.29, 133.71, 133.43, 129.52,129.41, 129.16, 129.13, 129.11, 128.98, 128.91, 128.64, 128.53, 127.71,127.49, 126.58, 120.16, 113.06, 100.86, 86.36, 85.38, 80.12, 78.06,71.84, 70.27, 69.94, 68.68, 67.89, 62.00, 54.94, 54.93, 54.84, 51.98,49.70, 45.44, 34.94, 34.60, 29.05, 28.97, 28.68, 28.63, 28.52, 26.09,25.03, 22.64, 21.78, 9.74, 7.13. LRMS calculated for C₇₈H₈₆N₈O₂₀1454.5958. found m/z 1455.2 (M+1)⁺, 1478.1 (M+23)^(Na+), 1453.2 (M−1)⁻.R_(f)=0.32 in 7% MeOH/DCM v/v

(7A)—Compound 6A (1.0 g, 0.69 mmol) was dissolved in acetonitrile (70ml) and HBTU (520 mg, 1.37 mmol) and DIEA (270 mg, 2.06 mmol) wereadded. The mixture was shaken for 5 minutes then LCAA-CPG (8.6 g, 510 Å,131 μmol/g) was added and shaken over night at room temperature. The CPGwas filtered and washed with 200 ml each of DCM, 20% MeOH/DCM v/v, anddiethyl ether then dried in vacuo. The CPG was shaken for 1 hour inacetic anhydride (12.5 ml), pyridine (37.5 ml), and triethylamine (0.5ml) before being washed again by the same conditions as before. Compound7A was dried in vacuo overnight and loading was measured with aspectrophotometer (73 μmol/g).

(8A)—Compound 5A (2.0 g, 1.48 mmol) was coevaporated with pyridine twiceand put under a strict argon atmosphere. DCM (15 ml) was added to theflask and cooled to 0° C. before the addition of 2-CyanoethylN,N,N′,N′-tetraisopropylphosphordiamidite (890 mg, 2.95 mmol). Themixture was stirred for 20 minutes then 4,5-Dicyanoimidazole (175 mg,1.48 mmol) was added to the reaction. The reaction was slowly warmed toroom temperature over night. The reaction was washed with saturatedbicarbonate and the aqueous layer extracted with DCM. The organic layerwas dried with Na₂SO₄, evaporated in vacuo to yield a pale yellow foam.The foam was loaded onto a pretreated manual column (ϕ=4.6×19) preparedwith 2% triethylamine/49% EtOAc/hexanes v/v. The impurities were elutedwith 80% EtOAc/hexanes v/v (8 CV) followed by 100% EtOAc (8 CV). TheEtOAc was then purged with 100% DCM (1 CV). 8A was eluted with 3%MeOH/DCM v/v (5 CV), and 6% MeOH/DCM v/v (5 CV), 8A was evaporated invacuo to yield 1.65 g (1.06 mmol, 72%) of the amidite as adiastereomeric mixture.

¹H NMR (400 MHz, DMSO-d₆) δ 11.40 (s, 1H), 8.42 (s, 1H), 8.05-7.85 (m,6H), 7.74-7.44 (m, 10H), 7.43-7.15 (m, 11H), 6.81 (q, J=8.8 Hz, 4H),5.95 (t, J=3.7 Hz, 1H), 5.75 (d, J=3.2 Hz, 1H), 5.36 (dd, J=11.1, 3.2Hz, 1H), 4.74 (d, J=8.5 Hz, 1H), 4.57-4.40 (m, 41-1), 4.30 (dq, J=29.0,9.1 Hz, 2H), 4.21-4.06 (m, 1H), 3.82-3.68 (m, 8H), 3.64-3.43 (m, 7H),3.30-3.19 (m, 4H), 3.04 (d, J=2.6 Hz, 3H), 3.00 (s, 3H), 2.97-2.86 (m,5H), 2.74 (t, J=5.6 Hz, 1H), 2.54 (t, J=5.9 Hz, 1H), 2.03 (s, 2H), 1.69(s, 3H), 1.56-1.36 (m, 6H), 1.34-1.04 (m, 19H), 0.95 (d, J=6.7 Hz, 3H),¹³C NMR (125 MHz, DMSO-d₆) δ 171.85, 169.52, 165.35, 165.30, 165.01,158.23, 158.19, 157.72, 157.41, 157.39, 149.96, 149.93, 144.86, 144.83,136.91, 136.66, 135.50, 135.49, 135.44, 135.40, 133.91, 133.64, 133.61,129.84, 129.80, 129.77, 129.71, 129.34, 129.32, 129.30, 129.17, 129.15,129.11, 128.84, 128.73, 127.91, 127.89, 127.79, 127.71, 126.83, 126.80,120.20, 119.00, 118.79, 113.25, 101.04, 85.79, 85.75, 85.26, 82.88,82.63, 80.07, 79.65, 72.01, 70.12, 68.88, 68.07, 63.50, 63.37, 62.18,58.78, 58.65, 58.21, 58.06, 55.15, 55.14, 55.12, 49.88, 45.74, 35.15,34.77, 29.30, 29.27, 29.24, 28.72, 26.39, 25.30, 25.28, 24.47, 24.44,24.42, 24.38, 24.34, 24.28, 22.83, 21.98, 21.21, 19.96, 19.91, 19.88,19.83, 18.96, 9.56. ³¹P NMR (160 MHz, DMSO-d₆) δ 154.02, 153.99. LRMScalculated for C₈₃H₉₉N₁₀O₁₈P 1554.6876, found m/z 1555.3 (M+1)⁺, 1578.3(M+23)^(Na+), 1555.4 (M−1)⁻, 1590.2 (M+35)^(Cl−). R_(f)=0.46 in 7%MeOH/DCM v/v.

Example 72

(4B)—Compound 3B (4.2 g, 4.75 mmol) was deprotected using the sameconditions as 3A to give compound 3B*.

LRMS calculated for C₃₇H₄₄N₆O₇ 684.3271. found m/z 685.2 (M+1)⁺, 707.2(M+23)^(Na+), 683.3 (M−1)⁻, 719.3 (M+35)^(Cl−). R_(f)=0.00 in 7%MeOH/DCM v/v

Compound 3B* was then treated with GalNAc(OBz)-C5-NHS ester (3.8 g, 5.22mmol) in the same manner as compound 3A*. 4.5 g of compound 4B wasobtained (3.43 mmol, 72%).

LRMS calculated for C₇₁H₇₇N₇O₁₇ 1299.5376, found m/z 1300.1 (M+1)⁺,1322.1 (M+23)^(Na+), 1298.2 (M−1)⁻, 1334.2 (M+35)^(Cl−). R_(f)=0.29 in7% MeOH/DCM v/v. ¹H NMR (500 MHz, DMSO-d₆) δ 10.91 (s, 1H), 10.65 (s,1H), 8.13 (d, J=9.3 Hz, 1H), 7.92 (1, J=6.5 Hz, 4H), 7.82 (d, J=7.5 Hz,2H), 7.76-7.67 (m, 3H), 7.64 (t, J=7.4 Hz, 1H), 7.57 (q, J=8.0 Hz, 3H),7.49 (t, J=7.7 Hz, 2H), 7.41-7.31 (m, 4H), 7.27 (t, J=7.6 Hz, 2H), 7.21(d, J 8.5 Hz, 5H), 6.89-6.81 (m, 4H), 6.75 (s, 2H), 5.75 (d, J=3.2 Hz,1H), 5.71 (d, J=4.7 Hz, 1H), 5.50 (d, J=6.0 Hz, 1H), 5.36 (s, 6H), 4.78(d, J=8.5 Hz, 1H), 4.60 (q, J=4.9 Hz, 1H), 4.45 (q, J=10.0, 8.2 Hz, 2H),4.38-4.24 (m, 2H), 4.03-3.95 (m, 2H), 3.79 (d, J 9.6 Hz, 1H), 3.72 (s,6H), 3.62-3.47 (m, 2H), 3.18 (ddd, J=44.2, 10.5, 3.7 Hz, 2H), 3.01-2.95(m, 2H), 2.06 (s, 2H), 1.70 (s, 3H), 1.56-1.41 (m, 6H), 1.35 (q, 1=6.6,6.1 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 171.74, 169.35, 165.18,165.12, 164.83, 158.00, 156.60, 153.94, 151.24, 144.70, 135.43, 134.92,133.74, 133.45, 129.61, 129.17, 129.15, 129.13, 129.00, 128.98, 128.94,128.68, 128.56, 127.75, 127.63, 126.63, 116.62, 113.10, 100.88, 86.80,85.54, 80.71, 77.77, 72.13, 71.91, 69.96, 69.62, 68.69, 67.88, 63.34,62.61, 62.04, 54.97, 54.90, 49.69, 34.95, 29.24, 29.12, 28.52, 26.27,25.21, 22.67, 21.81, 8.43.

(5B)—Compound 4B (4.3 g, 3.31 mmol) was protected in the same manner ascompound 4A to yield 3.0 g of compound 5B (2.21 mmol, 67%)

¹H NMR (400 MHz, DMSO-d₆) δ 11.37 (s, 1H), 10.46 (s, 1H), 8.51 (s, 1H),8.08 (d, J=9.3 Hz, 1H), 7.99-7.87 (m, 5H), 7.77 (t, J=5.5 Hz, 1H),7.73-7.52 (m, 7H), 7.48 (t, J=7.7 Hz, 2H), 7.37 (t, J=7.8 Hz, 2H), 7.31(d, J=7.3 Hz, 2H), 7.21 (dd, J=19.7, 8.2 Hz, 7H), 6.81 (dd, J=8.9, 2.4Hz, 4H), 5.82 (d, J=4.1 Hz, 1H), 5.74 (d, J=3.3 Hz, 1H), 5.52 (d, 1=5.8Hz, 1H), 5.36 (dd, J=11.1, 3.3 Hz, 1H), 4.81-4.67 (m, 2H), 4.44 (q,1=8.7, 7.5 Hz, 2H), 4.38-4.23 (m, 2H), 4.10 (1. J=5.3 Hz, 1H), 3.99 (q,J=4.8 Hz, 1H), 3.78 (d, J=9.6 Hz, 1H), 3.71 (s, 6H), 3.64-3.56 (m, 1H),3.50 (d, 1=9.7 Hz, 1H), 3.24-3.12 (m, 2H), 3.03 (d, J=7.3H, 10H), 2.05(s, 2H), 1.69 (s, 3H), 1.55-1.29 (m, 8H). ¹³C NMR (125 MHz, DMSO-d₆) δ171.73. 169.36, 165.19, 165.13, 164.84, 158.00, 157.99, 157.80, 157.57,157.22, 149.83, 144.70, 136.79, 135.46, 135.43, 133.74, 133.47, 133.45,129.58, 129.55, 129.17, 129.15, 129.14, 129.00, 128.98, 128.94, 128.68,128.56, 127.73, 127.60, 126.63, 119.77, 113.08, 100.89, 87.65, 85.46,80.73, 77.67, 72.05, 71.90, 69.96, 69.75, 68.71, 67.90, 63.22, 62.04,54.99, 54.97, 49.71, 34.98, 34.63, 29.22, 29.13, 28.54, 26.26, 25.20,22.67, 21.83, 8.38. LRMS calculated for C₇₄H₈₂N₈O₁₇ 1354.5798, found m/z1355.3 (M+1)⁺. 1377.2 (M+23)^(Na+), 1389.3 (M+35)^(Cl−). R_(f)=0.37 in7% MeOH/DCM v/v.

(6B)—Compound 5B (150 mg, 0.11 mmol) was treated with succinic anhydride(22 mg, 0.22 mmol) in the same manner as compound 5A to yield 120 mg ofcompound 6B (0.082 mmol, 75%).

¹H NMR (500 MHz, DMSO-d₆) δ 11.47 (s, 1H), 8.51 (s, 1H), 8.09 (d, J=9.3Hz, 1H), 7.99 (s, 1H), 7.92 (t, J=7.1 Hz, 4H), 7.76 (t, J=5.4 Hz, 1H),7.74-7.67 (m, 3H), 7.63 (t, J=7.4 Hz, 1H), 7.57 (q, J=8.2 Hz, 3H), 7.49(t, J=7.8 Hz, 2H), 7.38 (t, J=7.8 Hz, 2H), 7.29 (d, J=7.3 Hz, 2H),7.27-7.14 (m, 7H), 6.81 (d, J=7.9 Hz, 4H), 6.03 (d, J=2.7 Hz, 1H),6.00-5.97 (m, 1H), 5.76 (d, J=3.2 Hz, 1H), 5.37 (dd, J=11.1, 3.3 Hz,1H), 4.76 (d, J=8.5 Hz, 1H), 4.55-4.49 (m, 1H), 4.45 (q, J=9.9, 8.2 Hz,2H), 4.38-4.25 (m, 2H), 4.04-3.99 (m, 1H), 3.83-3.76 (m, 1H), 3.71 (s,6H), 3.54-3.41 (m, 2H), 3.40-3.26 (m, 6H), 3.14 (dd, J=10.7, 4.1 Hz,1H), 3.05 (s, 3H), 3.04-2.97 (m, 6H), 2.62-2.56 (m, 2H), 2.06 (s, 2H),1.70 (s, 3H), 1.56-1.45 (m, 4H), 1.34 (dd, J=19.5, 13.0 Hz, 4H), 1.17(s, 4H). ¹³C NMR (125 MHz, DMSO-d₆) δ 173.26, 172.01, 171.73, 171.46,169.36, 168.68, 165.18, 165.13, 164.84, 158.00, 157.90, 157.54, 157.36,149.37, 144.55, 137.17, 135.38, 135.31, 133.74, 133.48, 133.45, 129.54,129.51, 129.18, 129.15, 129.14, 129.01, 128.99, 128.94, 128.68, 128.56,127.73, 127.57, 126.63, 119.94, 113.06, 100.90, 86.10, 85.41, 80.53,75.77, 72.98, 71.88, 70.43, 69.96, 68.70, 67.90, 62.55, 62.02, 54.96,51.99, 49.71, 45.43, 34.97, 34.62, 33.24, 29.14, 29.10, 28.94, 28.84,28.54, 26.20, 25.18, 22.66, 22.59, 21.82, 21.13, 14.72, 10.03, 7.16,LRMS calculated for C₇₈H₈₆N₈O₂₀ 1454.5958. found m/z 1455.3 (M+1)⁺.1453.2 (M−1)⁻. R_(f)=0.43 in 5% MeOH/5% Et₃N/DCM v/v.

(7B)—Compound 6B (80 mg, 0.051 mmol) was loaded on to LCAA-CPG in thesame manner as 6A to yield 640 mg of CPG (69 μmol/g).

(8B)—Compound 5B (200 mg, 0.148 mmol) was coevaporated with ACN and thenwith pyridine. 5B was then phosphitylated in a similar manner as 5A toyield 80 mg of 8B as a diastereomeric mixture (0.051 mmol, 34.7%).

¹H NMR (400 MHz, DMSO-d₆) δ 11.41 (s, 1H), 8.47 (d, J=11.9 Hz, 1H),8.06-7.86 (m, 6H), 7.77-7.43 (m, 10H), 7.42-7.12 (m, 11H), 6.81 (dd,J=5.9, 2.7 Hz, 4H), 5.98 (dd, J 21.6, 3.6 Hz, 1H), 5.76 (d, J=3.2 Hz,1H), 5.38 (dd, J=11.1, 3.2 Hz, 1H), 5.07-4.92 (m, 1H), 4.75 (d, J=8.5Hz, 1H), 4.53-4.20 (m, 5H), 4.07-3.98 (m, 1H), 3.86-3.45 (m, 14H),3.41-3.13 (m, 8H), 3.02 (dd, J=10.2, 3.3 Hz, 8H), 2.74 (dd, J=12.3, 6.3Hz, 1H), 2.57 (t, J=5.9 Hz, 1H), 2.05 (s, 2H), 1.70 (s, 3H), 1.48 (d,J=27.8 Hz, 6H), 1.39-1.30 (m, 2H), 1.26-1.04 (in, 15H), 0.89 (d, J=6.7Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 171.89, 169.54, 165.36, 165.31,165.03, 158.21, 158.19, 157.82, 157.75, 157.71, 157.40, 149.88, 149.85,144.82, 137.15, 137.02, 135.57, 135.52, 135.50, 133.92, 133.65, 133.61,129.74, 129.35, 129.33, 129.32, 129.19, 129.17, 129.16, 129.11, 128.84,128.73, 127.92, 127.76, 126.83, 120.14, 120.01, 118.98, 118.80, 113.25,101.07, 85.74, 85.64, 72.02, 70.13, 68.92, 68.08, 62.19, 55.13, 55.11,49.90, 46.32, 45.78, 42.96, 42.86, 35.19, 34.78, 29.47, 29.41, 29.33,29.31, 28.75, 26.48, 25.44, 24.48, 24.43, 24.39, 24.37, 24.34, 24.05,23.99, 22.84, 22.04. ³¹P NMR (162 MHz, DMSO) δ 151.33, 150.99. LRMScalculated for C₈₃H₉₉N₁₀O₁₈P 1554.6876, found m/z 1555.3 (M+1)⁺, 1577.3(M+23)^(Na+), 1555.4 (M−1)⁻, 1591.2 (M+35)^(Cl−). R_(f)=0.46 in 7%MeOH/DCM v/v.

Example 73

(2)—Compound 1 (30.0 g, 106 mmol) was dissolved in DMF (300 ml) andcooled in an ice bath. NaH (3.86 g, 159 mmol) was added and the mixturestirred for 1 hour. N-(Bromohexyl)phthalimide (37.8 g, 122 mmol) wasthen added and the reaction heated to 70° C. over night. DMF wasevaporated in vacuo to a brown gum. The brown gum was dissolved in a 1to 1 mixture of DCM and WON for adsorbtion to silica gel. The solventmixture was removed in vacuo and the crude product was purified to yield8.10 g of 2 (15.83 mmol, 14.9%) as well as a mixture of 2 and its3′-O-alkylated regioisomer (23.45 g, 45.84 mmol, 43.2%). The total yieldof 2 and the regioisomer was 58%.

¹H NMR (400 MHz, DMSO-d₆) δ 7.95 (s, 1H), 7.89-7.77 (m, 4H), 6.78 (s,2H, D₂O exchangeable), 5.81 (d, J=6.7 Hz, 1H), 5.74 (s, 2H, D₂Oexchangeable), 5.51-5.43 (m, 1H, D₂O exchangeable), 5.08 (d, J=4.9 Hz,1H, D₂O exchangeable), 4.37 (dd, J=6.6, 5.0 Hz, 1H), 4.27-4.21 (m, 1H),3.92 (q, J=3.3 Hz, 1H), 3.67-3.59 (m, 1H), 3.52 (q, J=7.9, 7.0 Hz, 4H),3.34-3.30 (m, 1H), 1.45 (dt, J=38.0, 6.6 Hz, 4H), 1.24-1.12 (m, 4H), ¹³CNMR (100 MHz. DMSO-d₆) δ 167.90, 160.03, 156.21, 151.36, 136.06, 134.31,131.55, 122.95, 113.48, 86.18, 85.11, 80.52, 69.50, 69.12, 61.68, 54.88,28.94, 27.84, 26.00, 24.86. LRMS calculated for C-₂₄H₂₉N₇O₆ 511.5304.found m/z 512.2 (M+1)⁺, 534.2 (M+23)^(Na+), 546.2 (M+35)^(Cl−).R_(f)=0.50 in 5% MeOH/DCM v/v.

(3)—Compound 2 (100 mg, 0.195 mmol) was treated with1,3-Dichloro-1,1,13-tetraisopropyldisiloxane (90 mg, 1.5 eq) in pyridine(1 ml). After 3 days at room temperature, the reaction was quenched withMe011, washed with saturated bicarbonate, and extracted with DCM. Theorganic layer was dried with Na₂SO₄ and the crude product was purifiedwith silica gel chromatography to yield 120 mg of 3 (0.159 mmol, 82%).

¹H NMR (500 MHz, DMSO-d₆) δ 7.83 (dtd, J=8.7, 6.2, 3.4 Hz, 4H), 7.73 (s,1H), 6.76 (s, 2H), 5.73 (d, J=10.5 Hz, 3H), 4.52 (dd, J=8.6, 4.9 Hz,1H), 4.18 (d, J=4.8 Hz, 1H), 4.03 (dd, J=12.9, 2.1 Hz, 1H), 3.96-3.85(m, 2H), 3.78-3.70 (m, 1H), 3.66-3.58 (m, 1H), 3.53 (t, J=7.0 Hz, 2H),1.53 (dp, J=20.7, 6.8 Hz, 4H), 1.30 (ddt, J=44.2, 14.5, 7.2 Hz, 4H),1.07-0.95 (m, 28H). ¹³C NMR (125 MHz, DMSO-d₆) δ 167.89, 160.32, 156.12,150.97, 134.39, 134.33, 131.55, 122.95, 113.27, 86.56, 80.81, 80.63,70.46, 69.75, 60.23, 29.19, 27.95, 26.09, 25.25, 17.30, 17.18, 17.13,17.08, 16.96, 16.85, 16.81, 16.74, 12.75, 12.37, 12.23, 12.07. LRMScalculated for C₃₆H₅₅N₇O₇Si₂ 754.0356. found m/z 754.3 (M+1)⁺. 776.3(M+23)^(Na+), 753.3 (M−1)⁻, 788.2 (M+35)^(Cl−), R_(f)=0.31 in 5%MeOH/DCM v/v

(4)—Compound 3 (10 g, 13.26 mmol) was dissolved in pyridine (265 ml) andcooled in an EtOH ice bath. Isobutyryl chloride (1.55 g, 14.59 mmol) wasadded dropwise. The reaction mixture was stirred for 2 hours at −10° C.then 1 hour at room temperature. The reaction was quenched with MeOH andsolvents were removed in vacuo. The crude product was washed withsaturated bicarbonate and extracted with DCM. The organic layer wasdried with Na₂SO₄ and purified with silica gel chromatography (dinedwith EtoAc/Hexanes). Some N2 and N6 his protection occurred. 7.21 g ofpure 4 was obtained (8.75 mmol, 66%).

¹H NMR (400 MHz, DMSO-d₆) δ 9.73 (s, 1H), 8.02 (s, 1H), 7.83 (d, J=3.7Hz, 4H), 7.19 (s, 2H), 5.83 (s, 1H), 4.65 (dd, J=8.3, 5.2 Hz, 1H), 4.32(d, J=4.5 Hz, 1H), 4.15-4.07 (m, 1H), 3.97-3.85 (m, 2H), 3.81-3.72 (m,1H), 3.68-3.59 (m, 1H), 3.53 (t, J=6.9 Hz, 2H), 2.91-2.80 (m, 1H),1.62-1.45 (m, 4H), 1.41-1.21 (m, 4H), 1.20-1.07 (n, 3H), 1.06-0.83 (m,31H). ¹³C NMR (101 MHz, DMSO-d₆) δ 174.75, 167.82, 156.01, 152.89,149.44, 137.57, 134.27, 131.52, 122.89, 116.27, 87.34, 81.19, 80.98,70.49, 70.30, 60.83, 37.31, 34.01, 29.19, 27.92, 26.08, 25.16, 19.24,19.19, 17.28, 17.13, 16.97, 16.91, 16.88, 16.82, 12.53, 12.35, 12.12,12.04. LRMS calculated for C₄₀H₆₁N₇O₈Si₂ 823.4120, found m/z 824.2(M+1)⁺. 846.2 (M+23)^(Na+), 822.2 (M−1)⁻, 858.2 (M+35)^(Cl−). R_(f)=0.54in 100% EtOAc.

(5)—Compound 2 (500 mg, 0.977 mmol) was dissolved in DMSO (14 ml). PBS(0.1 M, 0.4 ml), and Tris (0.1 M, 12 ml). The pH was lowered to 7.33with 0.1 M HCl before the addition of adenosine deaminase (250 units, S.thermophilus recombinant in E. coli, CAS: 9026-93-1. SKU: 52544). Theformation of a white precipitate occurred shortly after the addition ofthe enzyme. The reaction proceeded for 3 days then was left anadditional 2 days but no more deamination occurred. The addition ofenzyme did not push the reaction forward. The pH of the reaction wasmonitored and adjusted to stay between 6.40 and 7.80. The whiteprecipitate was centrifuged down and the supernatant was decanted. Theprecipitate was transferred to a flask with pyridine and evaporated invacuo before being adsorbed to silica and purified with a manual column(ϕ=4.6×13.5). DMSO was elated with 100% EtOAc (2 CV), 4% WON in DCM v/v(1 CV) then 6% MeOH in DCM v/v (2 CV). The product 5 was then elutedwith 8% MeOH in DCM v/v (2.8 CV), 9% MeOH in DCM v/v (1.2 CV), andfinally 10% MeOH in DCM v/v (2.8 CV) to yield 290 mg of 5 (0.566 mmol,58%).

¹H NMR (400 MHz, DMSO-d₆) δ 10.61 (s, 1H, D20 exchangeable), 7.95 (s,1H), 7.88-7.78 (m, 4H), 6.44 (s, 2H, D₂O exchangeable), 5.76 (d, J=6.1Hz, 1H), 5.06 (s, 2H, D₂O exchangeable), 4.28-4.17 (m, 2H), 3.63-3.46(m, 5H), 3.38-3.28 (m, 3H), 1.46 (dt, J=37.2, 6.7 Hz, 4H), 1.19 (d,J=5.6 Hz, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.88, 156.61, 153.74,151.18, 135.36, 134.32, 131.55, 122.95, 116.57, 85.85, 84.42, 81.07,69.56, 68.88, 61.30, 37.26, 28.95, 27.83, 25.99, 24.86. LRMS calculatedfor C₂₄H₂₈N₆O₇ 512.2019, found m/z 513.1 (M+1)⁺. 511.0 (M−1)⁻, 547.0(M+35)^(Cl−), R_(f)=0.19 in 7% MeOH/DCM v/v.

(6)—Compound 4 (6.20 g, 7.52 mmol) was dissolved in water (45 ml) andglacial acetic acid (105 ml). NaNO₂ (4.15 g, 60.19 mmol) was then addedto the stirring mixture. After about 40 minutes, an additional 4.15 g ofNaNO₂ was added and stirred for 2 days at room temperature. Anadditional 2.1 g of NaNO₂ was added and stirred for an additional 2days. Starting material had been fully consumed according to TLC.Multiple spots were observed however corresponding to suspected silyldamage. The reaction was then diluted with water and extracted with DCM.The organic layer was washed with saturated bicarbonate then dried withNa₂SO₄. The product 6 was used as crude for the next desilylation step.

(7)—Compound 6 (7.52 mmol) was desilylated in THF (150 ml) with Et₃N.3HF(14.6 g, 90.28 mmol) over night. THF was removed in vacuo and the crudeproduct was adsorbed to silica then purified with silica gelchromatography (eluted with 5% MeOH/DCM v/v) to give a quantitativeyield. NMR showed ˜95% purity with ˜5% relating to a diamino nucleoside.

¹H NMR (400 MHz, DMSO-d₆) δ 12.04 (s, 1H), 11.64 (s, 1H), 8.27 (s, 1H),7.82 (p. J=4.4 Hz, 4H), 5.88 (d, J=6.6 Hz, 1H), 5.13 (d, J=4.7 Hz, 1H),5.05 (t, J=5.4 Hz, 1H), 4.28 (ddd, J=23.5, 6.8, 4.7 Hz, 2H), 3.92 (q,J=4.0 Hz, 1H), 3.63-3.50 (m, 3H), 3.46 (t, J=7.2 Hz, 2H), 3.35 (d, J=6.5Hz, 1H), 2.74 (p, J=6.9 Hz, 1H), 1.43 (dt, J=27.1, 6.5 Hz, 4H),1.25-1.13 (m, 4H), 1.09 (dd, J=6.7, 4.8 Hz, 6H). LRMS calculated forC₂₈H₃₄N₆O₈ 582.2438, found m/z 582.2 (M+1)⁺, 581.1 (M−1)⁻. 616.0(M+35)^(Cl−). R_(f)=0.41 in 7% MeOH/DCM v/v.

Table of Chemical Groups L96

Q150

L193

(T3gs)

(T3g)

(Tgs)

(Tg)

Q155

L199

Q160

L204

L200

Q156

Q154

L198

Q161

L207

L206

Q159

L203

L197

L208

L202

L201

L205

L223

L224

L221

(Uyg)

(Ayg)

(Cyg)

(Gyg)

Q151

1-14. (canceled)
 15. An oligonucleotide conjugate, wherein each ofnucleosides at the 7^(th), 8^(th) and 9^(th) position from the 3′-end ofthe oligonucleotide is conjugated to a carbohydrate-containing ligand atits 2′-position, wherein the conjugated nucleoside has the formula:

where the 5′ and 3′ ends are each attached to another nucleoside of theoligonucleotide or to a terminus; each occurrence of R⁶ is independentlya nucleobase; each occurrence of R⁷ is independently a linker; n is 1;each occurrence of R⁸ is independently selected from (a) —R²-R³, (b)R^(A), (c) R^(B), (d) R^(C), and (e) R^(D); each occurrence of R² isindependently absent or a spacer having two or more sites of attachmentfor the R³ groups, each occurrence of R^(A) is independently selectedfrom

each occurrence of R^(B) is independently selected from

R^(C) is selected from

R^(D) is selected from

R³ is a targeting monomer selected from


16. The oligonucleotide conjugate of claim 15, wherein theoligonucleotide is attached to R⁷ via a phosphate, phosphorothioate, ora combination thereof.
 17. The oligonucleotide conjugate of claim 15,wherein the oligonucleotide is attached to R⁷ via a cleavable group or anon-cleavable group.
 18. The oligonucleotide conjugate of claim 17,wherein R⁷ is selected from R^(E) and R^(F); R^(E) is selected from

R^(F) is selected from


19. The oligonucleotide conjugate of claim 15, wherein theoligonucleotide conjugate is a double stranded siRNA compound.
 20. Theoligonucleotide conjugate of claim 19, wherein the double strandedportion of a double stranded siRNA compound ranges 19 to 23 nucleotidespairs in length.
 21. The oligonucleotide conjugate of claim 15, whereinthe oligonucleotide conjugate further comprises one or more nucleotideshaving a 2′-F or 2′-OCH₃ group.